Protocols for the analysis of microRNA expression, biogenesis and function in immune cells

Abstract

MicroRNAs (miRNAs) are short (19–25 nucleotides) non-coding RNA molecules which target mRNAs to repress gene expression. It has been known that miRNAs play important roles in regulating many fundamental biological functions including cell differentiation, development, growth and metabolism. They are well conserved in eukaryotic cells and considered essential ancient elements for gene regulation. The miRNA gene is transcribed by an RNA polymerase II to generate a primary miRNA (Pri-miRNA), which is cleaved by a microprocessor complex in nucleus to generate a stem-loop structure known as pre-miRNA. Pre-miRNA is translocated to the cytoplasm and cleaved by DICER to form the mature miRNA. MiRNA can mediate mRNA degradation through loading of the microRNA to the RNA-induced silencing complex (RISC) and binding to the complementary sequences within a target mRNA to repress its translation by either mRNA degradation or translation inhibition or a combination of both. Because approximately 1900 miRNA genes have been reported from the human genome, many of which are associated with human diseases, the use of appropriate methods to study the expression of miRNA and its regulation under physiological and pathological conditions has become more and more important to the study of immune regulation. Similar to small interfering RNA (siRNA), the mechanism of miRNA mediated targeting has been applied to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze primary, precursor and mature miRNA expression and characterize their targets both in vitro and in vivo. Such studies will facilitate future novel drug design and development. This review is a summary of 6 basic protocols for miRNA analysis.

INTRODUCTION

MicroRNAs are small noncoding RNA molecules that play a key role in negatively regulating gene expression to control fundamental cellular processes (1). The number of miRbase entries has grown rapidly from 218 in 2002 to over 38,000 in 2018 (miRbase v. 22). More than 48,000 mature miRNAs have already been identified in 271 species with more than 1900 microRNAs in Homo sapiens (miRbase, release 22). The rapid growth in identifying new microRNAs has been associated with links to numerous human diseases, such as cancer, autoimmune disease, metabolic disorders, obesity, renal disease and viral diseases (1–6). It is estimated that in human, ~60% of the transcriptome is under miRNA regulation (7, 8). Many miRNAs are evolutionarily conserved, indicating they have important biological functions.

miRNAs are initially transcribed as primary transcripts which are subsequently cleaved by the double-stranded RNA-specific endoribonucleases Drosha and Dicer, to generate ~70 nucleotide (nt) long precursor (hairpin) miRNAs and ~20 nt long miRNA duplexes (9), respectively. One of the two RNA strands become a functional miRNA and retains Argonaut protein binding, while the other is released and degraded (10, 11). Traditional mature miRNAs post-transcriptionally regulate gene expression by imperfectly binding to target mRNAs in association with a multiprotein RNA-induced silencing complex (RISC), resulting in either mRNA degradation or translational repression (12).

The clinical applications of miRNA are now just beginning to draw attention. First, they were tested for potential use as biomarkers for diagnosis, subtyping, and estimation of disease progression, predominantly in cancer. Some miRNA appear in blood, because of their resistance to degradation. Cancer patients have been reported to have elevated cancer-specific miRNAs in their blood or serum, because removal of the primary tumor results in decreased levels of these circulating miRNAs (2). For example, increased levels of circulating miR-195 was found to be breast cancer-specific, allowing it to be used to distinguish breast cancer from other cancers (13). Second, they have been tested as reporters of drug resistance (14, 15). Third, the most important and challenging use of miRNA mechanisms have been to develop miRNA-based therapeutics (16). Indeed, miRNA-based therapeutics have been approved for clinical use to treat a type of rare peripheral nerve disease (polyneuropathy), caused by hereditary transthyretin-mediated amyloidosis (hATTR) in adult patients (17). Recently, new subsets of microRNAs were identified as non-canonical microRNAs including snoRNAs, introns, and tRNAs (18). Although these are similar to canonical miRNAs in term of function, non-canonical microRNAs are synthesized by distinct biogenesis pathways. They have been reported to play important roles in the human immune response as well as stem cell proliferation (18, 19). However, many of their functions remain unknown.

It is thus of interest to analyze the most common forms of human miRNAs-- canonical miRNAs and determine their expression and function both in vivo and in vitro. In the following protocols, we have included methods for microRNA isolation and small RNA or microRNA sequencing to allow expression profiling of microRNA during immune regulation. We have also included protocols for mapping Drosha cleavage sites involved in microRNA biogenesis. In addition, Electrophoretic Mobility Shift Assays (EMSA) provide methods to identify the proteins in the miRNA ribonucleoprotein complexes termed miRNPs. Finally, we have described the overexpression or inhibition of microRNAs as useful tools for their functional analysis. Topics include:

1. Next-generation sequencing to a) identify new miRNAs and their precursors or b) compare the abundancy of known miRNAs between different cells, tissues or other biological samples. c) obtain general profiles of microRNA expression.

2. Analysis of primary, precursor miRNA and mature microRNA expression by RT-qPCR.

3. Use of non-radioactive digoxigenin-labeled RNA probes to identify Pri-miRNA, precursors and mature miRNA.

4. RACE protocols for mapping Pri-miRNA and precursor miRNA cleavage sites.

5. Analysis of miRNP complexes in vitro.

6. Use of miRNA mimics or inhibitors for miRNA functional analysis in cells ex vivo.

Basic Protocol 1. MicroRNA extraction, isolation, amplification, cDNA preparation, and sequencing from human B Cells

Next-generation small RNA sequencing does not require known sequencing data to design specific probes typical for DNA microarray or the cloning methods required for Sanger sequencing (20). Several sequencing platforms are available for miRNA-sequencing, including the Solexa/Illumina Genome Analyzer (21). This sequencing method provides a new tool for the discovery of thousands of small RNAs, miRNAs and other small noncoding RNAs sequences. It can read the differential expression of all types of small RNAs, and map the individual reads to the proper reference genome. Unlike the traditional Sanger sequencing method, deep sequencing allows the identification of SNPs in closely related RNAs and discover 5’- or 3’- end variations in addition to measuring the absolute abundance of miRNAs (22). Furthermore, it allows the identification of novel small RNAs, since it is not restricted by known sequence information.

Total RNA isolation kits from different companies have been compared for miRNA analysis in terms of quantity, quality and high recovery of miRNAs from cells or tissues (23). One study found that phenol: chloroform phase separation combined with silica column-based solid extraction methods were preferable. (miRVana microRNA isolation Kit, with phenol). This kit allows efficient isolation of small RNA-containing total RNA and is ideal for miRNA, siRNA, shRNA and snRNA analysis.

Materials

Solexa/illumina Genome Analyzer

miRVana microRNA isolation kit, with phenol AM1560

• miRNA washing solution 1

• miRNA wash solution 2/3

• Filter Cartridges

• Lysis/binding buffer

• miRNA homogenate additive

• Acid-Phenol: Chloroform

• miRNA Wash Solution 1

• miRNA Wash Solution 2/3

100% Ethanol (Room temperature)

75% Ethanol (Room temperature)

RNase free water

15% Novex TBE-urea PAGE Gel [1x TBE, 7M urea, 15% acrylamide (19:1 acryl:bis-acryl)] Cat.# EC6885BOX

6% Novex TBE-urea PAGE Gel Cat.# EC6865BOX

5x Novex TBE running (Tris/Borate/EDTA) buffer, ThermoFisher, Cat.#LC6675)

1x Novex TBE running buffer (1:5 dilution from 5x Novex running buffer)

Small RNA Sample Prep kit, catalog# FC-102–1009, Illumina

• RNase OUT part#1000560

• UltraPure water (store at 4°C), part# 1000467

• 10x Gel Elution Buffer, part# 1000571

• 1x Gel Elution Buffer (1:10 dilution from 10x Gel Elution Buffer with

• Ultrapure water)

• Small RNA (SRA) Ladder, part #1001665

• Small RNA (SRA) Gel Loading Dye, Part# 1001661

• Glycogen, part # 1001664

• SRA 0.3 M NaCl, part # 1000573

• Spin X cellulose acetate filters, part # 1001673

• Phusion^™ Polymerase (Finnzymes Oy), part # 1000584

• 5x Phusion HF Buffer (Finnzymes Oy), part # 1000585

• Primer GX1, part # 1000591

• Primer GX2, part # 1000592

• 25 bp ladder, part # 1001662

• SRA RT Primer, part # 1000597

• T4 RNA ligase, part# 1000587

• 10x T4 RNA ligase buffer, part# 1000588

• Resuspension Buffer, part# 1001338

• 25bp ladder, part# 1001662

• 25 mM dNTP Mix, part # 1001663

• 6x DNA loading dye, part# 1003029

• SRA 5’ Adapter, part# 1000595

• SRA 3’ Adapter, part# 1000596

Ultrapure ethidium bromide 10 mg/ml, ThermoFisher, Cat.# 15585011

0.5µg/ml of ethidium bromide in 1x TBE buffer

Human B Cells (Isolated from human normal volunteer’s PBMC. It is an EBV

Infected B cell line provided by Dr. Jefferey Cohen at NIH)

1.5-ml microcentrifuge tubes

Clean scalpels

21-gauge needles

SuperScript II Reverse Transcriptase with 100 mM DTT and 5x First Strand

Buffer Cat #18064014

100 mM dithiothreitol (DTT) (ThermoFisher, Cat.# R0861)

Sterile RNase-free PCR tubes

Stacked tubes (Made by puncturing the bottom of a sterile, Rnease-free, 0.5 ml microcentrifuge 4–5 times with a 21-gauge needle and placing it into a sterile, round-bottom, nuclease-free, 2 ml microtube)

Equipments

• Vortex

• Benchtop microcentrifuge

• 4°C microcentrifuge

• Dark Reader transilluminator (Clare Chemical Research, Part# D195M)

• Electrophoresis power supply

• Benchmark Roto-Therm^™ Incubated Tube Rotators (The Lab Depot, Inc.

• Cat.#H2024)

• Heat block

• Savant speed vac.

• Thermal cycler (Bio-Rad)

XCell Sure Lock Mini-Cell electrophoresis unit (Invitrogen, Part#E10001)

Total RNA isolation

Basic protocol 1 steps 2–18 are conducted using the miRVana microRNA isolation kit, with phenol. Typically, this kit can process mammalian cells, tissues, viral samples, bacteria, yeast and plant cells. This kit allows efficient isolations of small RNA enriched total RNA samples.

• 1Samples of 10^6 purified human B cells are collected and washed in 20 mL cold PBS. Disrupt the cells by adding 300–600 μl Lysis/Binding buffer followed by addition of 1/10 volume (30–60 ul) of miRNA homogenate additive to the cell lysate. Mix well by inverting the tube several times.
• 2Incubate on ice for 10 min., add an equal amount (300–600 uL) of Acid-Phenol: Chloroform to the mixture.
• 3Vortex samples for 30~60 second and then centrifuge for 5 minutes at maximum speed (10,000x g) at 4°C to separate the aqueous and organic phases. Carefully remove the aqueous (upper) phase by pipette without disturbing the lower phase and transfer to a fresh tube.
• 4Add 1.25 volumes of 100% ethanol to the aqueous phase (e.g. for 300µL sample, add 375 µL ethanol).
• 5Place a filter cartridge from the microRNA isolation kit into one of the collection tubes and pipet the lysate/ethanol mixture onto the filter cartridge. For each filter cartridge, a maximum of 700 µL of sample can be applied each time. If the sample volume is larger than this, repeatedly apply the mixture to the same filter.
• 6Centrifuge the samples at RCF 10,000 x g (typically 10,000 rpm). Higher speed may damage the filter. Alternatively, vacuum pressure can be used to pass samples through the filter.
• 7Discard the flow-through and repeat until all the lysate/ethanol mixture is through the filter.
• 8Spin the filter cartridge with 700 µL miRNA Wash Solution 1 (working solution) once and 500 µL Wash Solution 2/3 (working solution) twice at 15,000rpm for 1 minute. After discarding the flow-through from the last wash, spin the filter cartridge assembly for an additional 1 minute to remove residual fluid from the filter.
• 9Transfer the filter cartridge into a fresh collection tube. Add 100 μl of pre-heated (95°C) RNAse-free water to the center of the filter and spin for ~20–30 sec at 15,000rpm for 1 minute to recover the RNA. Store the RNA at −20°C or below (up to 2 years).
• 10To enrich the small RNAs, add 1/3 volume of 100% ethanol to the aqueous phase recovered from the organic extraction (e.g. Add 33 µL 100% ethanol to 100 µL aqueous phase). Mix well by vortexing.
• 11Apply the mixture onto a filter cartridge with the collection tube (a maximum of 700 µL can be loaded to a filter cartridge at a time). Centrifuge for ~15 sec at 10,000 x g to collect the filtrate. If the sample’s volume is greater than 700 µL, transfer the flow-through to a fresh tube and repeat this step a few times to the same filter cartridge until all sample is through the filter and collected. Estimate the total volume of the filtrate.
• 12Add 100% ethanol to the filtrate such that the final ratio of ethanol:filtrate would be 2:3 (e.g. add 266 µL 100% ethanol to 400 µL of recovered filtrate). Gently agitate this mixture.
• 13Apply the mixture onto a second filter cartridge (a maximum volume is again 700 µL; if greater than 700 µL, repeat this step a few times as described above). Centrifuge at 10,000 x g for ~15 sec to pass the mixture through the filter.
• 14Discard the flow-through and re-use the collection tube for the washing steps.
• 15Add 700 µL miRNA wash solution 1 to the filter cartridge and centrifuge for ~5–10 sec to pass the solution through the filter. Discard the flow-through.
• 16Add 500 µL wash solution 2/3 to wash the filter cartridge as in the previous step.
• 17Repeat with a second 500 µL aliquot of wash solution 2/3. Discard the flow through and spin the assembly for 1min to remove residual fluid from the filter.
• 18Transfer the filter cartridge into a fresh collection tube. Apply 35 µL of pre-heated (95°C) Rnase-free water to the center of the filter, and close the cap. Spin for ~20–30 sec at maximum speed to recover the RNA. Store the RNA at −20°C or below.

We typically analyze the quality of RNA extracted with this method by using gel electrophoretic analysis (24). High quality RNA will demonstrate a 28S-rRNA band at 4.5 kb and an 18S-rRNA band at 1.9 kb on a denaturing agarose gel stained with 10 mg/ml ethidium bromide (EtBr). If the 28S rRNA band is approximately twice as intense as the 18S rRNA band, it is a good indication that the RNA is completely intact. Alternatively, total RNA integrity number (RIN, which is an algorithm used to determine the quality of RNA, can also be verified by an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than 8 (25).

Isolation of Small RNA (Small RNA Sample Prep kit, Illumina)

This process purifies small RNA from about 10 ug of total RNA by a separation based on nucleotide length, recovering small RNA (18–30 nucleotides) from a band in denaturing gels that corresponds to the nucleotide length of interest. It is not recommended that multiple samples be purified on a single gel due to the risk of cross-contamination of libraries. This applies to each gel purification step in this protocol.

• 19Warm the gel by pre-running a 15% TBE-urea PAGE Gel in 1xTBE running buffer at 200V for 15–30 minutes. While the gel is running, mix 2 μl of SRA ladder with 2 μl of SRA gel loading dye in a sterile RNase-free PCR tube.
• 20Mix 10 μl (10μg) of total RNA (from Basic Protocol 1 step 18) with 10 μl of SRA gel loading dye in a sterile, RNase-free microcentrifuge tube.
• 21Heat the sample and ladder tubes at 65°C in a thermal cycler for 5 min to denature the RNA, then place the tubes in ice. Spin PCR product in microcentrifuge tubes briefly to collect the entire sample in each tube.
• 22Combine 4 μl SRA RNA ladder and 20 μl samples of RNA, then load it on the same gel across several lanes. Run the gel at 200V for 1 hour.
• 23While the gel is running, prepare the stacked tubes by puncturing the bottom of a sterile, Rnease-free, 0.5 ml microcentrifuge 4–5 times with a 21-gauge needle and placing it into a sterile, round-bottom, nuclease-free, 2 ml microtube.
• 24Remove the gel from the apparatus and stain it with 0.5µg/ml Ethidium Bromide in 1x TBE buffer in a clean container for 2 minutes.
• 25View the gel on a Dark Reader transilluminator to avoid being exposed to the UV light. The SRA ladder ranges from 20–100 bases in 10 base increments. Excise the gel band corresponding to an 18–30 nucleotide length (by referring to the RNA ladder) using a clean scalpel and transfer it into a 0.5 ml microcentrifuge stacked tube prepared in the previous step.
• 26Centrifuge the stacked tubes at full speed for 2 minutes at room temperature to move the gel through the holes into the 2 ml tube.
• 27Extract RNA from the gel debris by adding 300 μl of SRA 0.3M NaCl and gently rotating the tube at room temperature for 4 hours.
• 28Transfer the eluate and gel debris to the top of a Spin-X cellulose acetate filter. Centrifuge the filter at maximum speed for 2 minutes at room temperature. Add 3 μl of glycogen and 750 μl of room temperature 100% ethanol to the tube.
• 29Incubate at −80°C for 30 minutes followed by immediate centrifugation of the spin-X tube on a 4°C microcentrifuge at 14,000 rpm for 25 minutes to precipitate the RNA.
• 30Remove the supernatant and discard it. Wash the pellet with 750 μl of room temperature 75% ethanol. Remove the supernatant and discard it.
• 31Allow the RNA pellet to air dry and resuspend the pellet in 5.7 μl of Rnase-free water.

cDNA library preparation - Ligation of 3’ and 5’ Adapters to isolated small RNAs

This process ligates adapters to the 3’ and 5’ ends of the isolated small RNAs. It uses 5’phosphate and 3’hydroxyl groups on the small RNA, which allows the ligation with 5’ and 3’ single stranded adapters by RNA ligases. These adapters can also work as reverse transcription primer and PCR binding sites.

• 32Mix the 5.7 μl purified small RNA in water with 1.3μl of SRA 5’-RNA adaptors, 1μL of 10x T4 RNA ligase buffer, 1μL of RNase inhibitor and 1μL of T4 RNA ligase.
• 33Incubate the mixture at 20 °C for 6 hours in a thermal cycler followed by storage overnight at 4°C. The next morning, stop the reaction by adding 10 μl of SRA gel loading dye.
• 34Run the ligated RNA on a gel as described above (steps 19–24). Use a 15% TBE-urea PAGE Gel and cut out a band of gel corresponding to 40–60 nucleotides using the same elution and ethanol precipitation steps as described above (steps 25–29).
• 35Resuspend purified small RNA in 6.4 μl of ultrapure water.
• 36Mix the 6.4 μl of RNA solution with 0.6 μl of SRA 3’-RNA adaptor, 1 μl of 10x T4 RNA ligase buffer, 1 μl of RNase inhibitor, and 1 μl of T4 RNA ligase.
• 37Incubate at 20°C for 6 hours in a thermal cycler and hold overnight at 4°C.
• 38Stop the reaction by adding 10 μl of SRA gel loading dye.
• 39Purify the RNA by following the same procedure as described above (steps 19–24) to cut out a band of gel corresponding to the 70–90 nucleotide length and recover the RNA from the gel as describe above (steps 25–29).
• 40Resuspend the RNA pellet in 4.5 μl of ultrapure water.

Reverse transcription and PCR amplification

Reverse transcription and PCR are used to make cDNA constructs based on the small RNA ligated with 5’ and 3’ adapters. This protocol selectively adds adapter molecules on both ends of RNA fragments followed by a PCR performed with two primers that anneal to the ends of the adapters.

• 41Dissolve the purified 5’ and 3’ ligated small RNA in 4.5 μl of ultrapure water and mix with 0.5 μl of SRA adaptor RT primer.
• 42Heat the mixture on a thermal cycler at 65°C for 10 minutes and immediately transfer onto ice.
• 43Prepare a master mix of 12.5mM Deoxyribonucleotide triphosphate (dNTP) in a separate, sterile, RNase-free, 200 μl PCR tube. Mix 0.5μL ultrapure water and 0.5 μl of 25 mM dNTP Mix for each sample. Multiply the volume by the number of samples being prepared. Make 10% extra reagent if there are multiple samples.
• 44Preheat the PCR thermal cycler to 48°C. for each sample, mix 2 μl of 5x first strand buffer, 0.5 μl of 12.5 mM dNTP mix, 1 μl of 100 mM dithiothreitol (DTT) and 0.5 μl of RNase inhibitor. Multiply each volume by the number of samples being prepared. Prepare 10% extra reagent if there are multiple samples.
• 45Add 4 μl of the mix to the 5 μl cooled primer-annealed template material in step 41.
• 46Heat the sample at 48°C on a thermal cycler for 3 minutes and add 1 μl SuperScript II Reverse Transcriptase followed by incubating the sample at 44°C for 1 hour to perform the reverse transcription.
• 47To prepare the PCR master mix, premix 28 μl of ultrapure water, 10 μl of 5x Phusion HF buffer, 0.5 μl of Primer GX1, 0.5 μl of primer GX2, 0.5 μl of 25 mM dNTP mix and 0.5 μl of Phusion DNA polymerase.
• 48Mix 10 μl of reverse-transcribed cDNAs and 40 μl of PCR master mix in a sterile, nuclease-free 200 μl PCR tube and perform the PCR reaction using following protocol: (1) 30 seconds at 98°C (2) 15 cycles of 10 seconds at 98°C, 30 seconds at 60°C, 15 seconds at 72°C (3) 72°C 10 minutes (4) hold at 4°C.
• 49Mix 1 μl of 25 bp Ladder with 1 μl of 6x DNA loading dye.
• 50Mix 50 μl of amplified cDNA with 10 μl of 6x DNA loading dye. Load 2 μl of mixed 25 bp ladder and load two wells with 30 μl each of mixed amplified cDNA sample on the 6% PAGE Gel. Run the gel for 30–35 minutes at 200 V.
• 51Dilute the 10X Gel elution buffer to 10 ml of 1X gel elution buffer with ultrapure water.
• 52Prepare the stacked tubes as described in step 23.
• 53Remove the gel from the apparatus. Stain the gel with 20 ml of 0.5µg/ml of ethidium bromide in 1x TBE buffer in a clean container for 2–3 minutes. View the gel on a Dark Reader Transilluminator or a UV transilluminator.
• 54Use a clean scalpel, cut out approximately a 92-bp band in the sample lanes. Transfer the band to the stacked tube prepared in step 51.
• 55Centrifuge the stacked tubes at maximum speed for 2 minutes at room temperature to move the gel through the holes into the 2 ml tube.
• 56Add 100 μl of 1x gel elution buffer to the gel debris in the 2 ml tube.
• 57Elute the DNA by rotating the tube gently at room temperature for 2 hours.
• 58Transfer the elute and the gel debris to the top of a Spin-X cellulose acetate filter.
• 59Centrifuge the filter at 15,000 rpm for 2 minutes at room temperature.
• 60Add 1 μl of glycogen, 10 μl of 3M NaOAc, and 325 μl of −15°C to −25°C 100% ethanol to the Spin-X tube.
• 61Immediately centrifuge the Spin-X tube for 20 minutes at room temperature.
• 62Remove the supernatant and wash the pellet with 500 μl of room temperature 70% ethanol.
• 63Remove and discard the supernatant and dry the pellet using a speed vac or simply leave it open to air at room temperature for 15 minutes.
• 64Resuspend the DNA pellet in 10 μl of resuspension buffer. load 1 μl of the product on a NanoDrop^™ Spectrophotometer to measure the concentration and purity of the sample. Or clone 1 μl of the product into TOPO cloning vector (Invitrogen) and sequence cloned insertions by conventional sequencing technology

  The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A ratio of ~1.8 is generally accepted as “pure” for DNA. If the ratio is lower, it may indicate the presence of protein or other contamination that absorb strongly at or near 280 nm. 260/230 ratio is used as a secondary measure of nucleic acid purity. The expected 260/230 values for a “pure” sample are in the range of 2.0–2.2. If the ratio is considerably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.

• 65Send purified cDNA samples to a suitable core sequencing center such as the NIH intramural sequencing center (NISC) for small RNA sequencing by an Illumina Genome Analyzer using the recently revised protocols for Illumina sequencing (26). The amount of starting material is usually >5 µg but can go as low as 1 µg.
• 66Typical Illumina sequencing of this library would collect 5 million reads per sample, length of each being 50 to 75 bases. Raw sequence data in FASTQ file format would adapter-trimmed to remove any amplification or sequencing adapters leaving only the biological nucleotide sequence. The biological sequence will include miRNA variants with exact match to reference, some with mutations, and represent mature as well as not fully processed sequences, depending on the individual miRNA identity and biological condition.
• 67Annotation of the sequences involves finding the closes reference match in databases, typically miR-base, followed by search of non-coding RNAs in the genome build for those sequences not matching any in miR-base.
• 68Sequences then can be grouped according to a common mature reference miRNA sequence and reported as total read counts for a given mature miRNA, taking the sum of mature reads and all forms of variants that can be attributed. [this is the approach used by commercial packages such as CLC-bio and StrandNGS]
• 69An alternative approach suggested by ENCODE involves adapter trimming followed by mapping to microRNA annotated genomic locations using the STAR aligner. It uses STAR to count the unique matches to each annotated miRNA and provides a signal track suitable for visual inspection of the coverage at any genomic location. (https://www.encodeproject.org/microrna/microrna-seq/)
• 70Biological replicates are necessary to confirm the expression values. At least two replicates are advised if a simple qualitative analysis of presence/absence or crude quantitative estimates are required. Alternatively, a quantitative estimate of miRNA expression differences or expression level confidence estimates are required, at least three biological replicates are necessary.
• 71Statistical analysis of miRNA count data is remarkably different from mRNA due to the nature of the transcript abundance distribution, for micro RNA it is much more skewed to very few transcripts with detectible or large expression levels, the bulk of miRNA genes with little to no none. Whereas a typical mRNA-seq expression estimate for protein coding genes would quantify 1/3 to 1/2 of all empirically validated genes in the reference genome. Having biological replicates from at least three independent experiments and relatively deep sequencing with at least 5 million reads per sample, will improve the efficiency of sample to sample normalization and precision of differential expression estimates.

Basic Protocol 2. Analysis of primary, precursor and mature miRNA expression by qRT-PCR

Primary miRNAs are transcribed in the nucleus by RNA polymerase II, which is further cleaved by Drosha (RNase III enzyme) to produces a stem-loop structure of about 70 nt as a pre-miRNA hairpin. Pre-miRNA is then exported by Exportin-5 through the nuclear pore complex to the cytoplasm where it is recognized and cleaved by Dicer to produce a short double-stranded miRNA. The double strained miRNA is loaded to AGO2 protein and unwinded. One of the strains is degraded (the passenger strain), and the other strain becomes the mature miRNA. AGO2 bounded mature miRNA is recruited to the RISC complex to mediate mRNA targeting and decay. Since the expression level of mature miRNA can be regulated at any step in the miRNA biogenesis and maturation pathway (27), it is necessary to use proper tools for analyzing miRNA at different steps during its synthesis and maturation.

The method described here allows the quantification of the expression of miRNAs using a small amount of RNA sample. It was difficult to quantitate the small miRNA fragments by qRT-PCR due to their small length and sequence similarity, however, a number of miRNAs qRT-PCR methods have been introduced since 2004 (28). TaqMan qRT-PCR and SYBR Green are the most common methods used for this analysis. TaqMan probes increased the specificity of the qPCR. A minor groove binder (MGB) conjugated Taqman probe can increase its binding activity to the target sequence. A standard Taqman microRNA assays use target specific stem-loop reverse transcription primers to extend the length of miRNA template followed by Taqman assay real time PCR (29). Since a mature miRNA is a part of precursor and primary miRNAs, it is important to design specific primers to distinguish each type of microRNA and its isoforms by qRT-PCR.

For each qRT-PCR, reactions are usually done in triplicate to reduce effects from technical variability. A negative control without template should be included and a dissociation curve should be calculated to determine if there is any nonspecific primer interactions. To quantitate mature miRNA expression, two qRT-PCR methods were compared (30). The first method (Taqman) utilizes stem-loop RT primers to produce cDNA of specific miRNAs. The second method (SYBR) uses a modified oligo(dT) primers to reverse transcribe all transcripts within an RNA sample, therefore allowing miRNA and mRNA to be analyzed in the same RT reaction. There were no significant differences (P>0.05) in miRNA expression between the stem-loop and modified oligo (dT) RT methods according to one review, suggesting both methods are statistically equivalent (30). Since the oligo (dT) method requires only a single RT reaction to reverse both miRNA and mRNA and it is less time consuming, the protocol described here will focus on the oligo(dT) method.

Materials

Primers for mature miRNA or pre-miRNA or pri-miRNA - Human MicroRNA Assay kit (Qiagen)

miScript II RT kit (Qiagen, Cat.# 218161

• miScript Reverse Transcriptase Mix (optimized blend of poly(A)

• polymerase and reverse transcriptase)

• 10x miScript nucleics Mix

• 5x miScript HiSpec buffer

• 5x miScript HiFlex Buffer

• miScript SYBR Green PCR kit (Qiagen, Cat.# 218073)

• 2x QuantiTect SYBR Green PCR Master Mix

• 10x miScript universal primer (included in the kit)

Hs_miR-15b miScript Primer assay (Qiagen, Cat.# MS00003185)

Hs_RNU6 miScript Primer Assay (Qiagen, Cat.# MS00033740)

Tabletop centrifuge with swinging bucket

A Real Time PCR instrument (Bio-Rad, IQ5)

Unskirted 96-Well PCR Plates (Bio-Rad, MLL9601)

Microseal ‘B’ PCR Plate Sealing Film, adhesive, optical (Bio-Rad, msb1001)

RNase free water

Reverse transcription of total RNA to generate cDNA

• 1Conduct basic protocol 1 steps 1–18 on 10⁶ human B cells.
• 2Resuspend 100 ng RNA in 5 μl of RNase-treated water and perform the reverse transcription using miScript II RT kit.
• 3Thaw 10x miScript nucleic mix and either 5x miScript HiSpec buffer (for mature miRNA quantification) or 5x miScript HiFlex Buffer (for precursor miRNA detection) at room temperature (15–25°C).
• 4Prepare the reverse-transcription master mix on ice containing 4 μl of miScript RT buffer (5x), 2 μl 10x miScript nucleic mix, 2 μl miScript reverse transcriptase mix and 7 μl of DEPC-treated water. Add 15 μl of master mix to 5 μl of total RNA from step 1 and incubate at 37°C for 1 hour followed by inactivation of the enzyme by heating to 95°C for an additional 5 minutes and placing on ice.
• 5Dilute the cDNA samples from step 4 to 1:10 by adding 180μl of Rnase-free water to 20μl of the cDNA sample.

For example, in this protocol, we used RNA samples isolated from purified human B cells with or without EBV infection. We are comparing the expression of hsa-mir15b under both conditions using human snU6 as a control. After reverse transcription, you should have two cDNA samples in 20 μl.

Quantitative real time PCR reactions

• 6Thaw all reagents and the cDNA templates prior to preparing real-time PCR reaction mixes. Gently mix the contents in each tube and briefly centrifuge.
• 7To detect mature miRNA only or mature miRNA and other noncoding RNA, prepare a miRNA master mix with 1 μl of 10x miScript universal primer, 1 μl of 10x miScript specific miRNA primer (hsa-miR-15b miScript Primer assay or Hs-RNU6 miScript Primer Assay), 5 μl of 2x Quantitect SYBR Green PCR Master Mix and 2 μl of DEPC-treated water.
• 8Add 9 μl of master mix to 1 μl of sample (diluted cDNA from step 4). The volume of diluted cDNA should not exceed 10% of the final reaction volume. The final concentration of cDNA should be 50 pg - 3 ng per reaction in each well. Seal the plate with an optical adhesive cover, and centrifuge at 1,000 x g for 20 min in a swinging bucket plate holder.
• 9To detect precursor miRNA, prepare a master mix with 5 μl of 2x QuantiTect SYBR Green PCR Master Mix, 1 μl of 10x miScript Precursor Assay (containing both a forward and a reverse primer) and 3 μl of DEPC-treated water.
• 10Add 9 μl of master mix to 1 μl of sample (diluted cDNA from step4. The volume of diluted cDNA should not exceed 10% of the final reaction volume. The final concentration of cDNA should be 10–20 ng per reaction.) in each well, seal the plate with an optical adhesive cover, and centrifuge at 1,000 x g for 20 min in a swinging bucket plate holder.
• 11Load the plate into a real time PCR instrument (Bio-Rad, IQ5) and run the reaction with the following program: Initiate activation of the HotStartTaq DNA Polymerase by a hot start at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 15s, annealing at 55°C for 30s and extension at 70 °C for 30s. (Fig.1a)

Fig.1.

Measurement of the expression of hsa-miR-15b-5p by reverse transcription and quantitative real time PCR (RT-qPCR). a) Reverse transcribed cDNAs are used for a quantitative real time PCR analysis (qPCR). An amplification chart of qPCR for hsa-miR-15b-5p (blue) and U6 (internal control, pink). b) RNAs isolated from RNA immunoprecipitation (RIP) complexes by an anti-pan Ago monoclonal antibody and an isotype control were reverse transcribed into cDNA. The enrichment of hsa-miR-15b-5p from both were detected by qPCR. A table of threshold cycles (Ct) for the amplification of hsa-miR-15b-5p under both conditions. ΔCT=Ct_Ago-Ct_Iso. c) Relative fold enrichment of hsa-miR-15b-5p in isotype IgG RIP vs. anti-pan-Ago RIP. The enrichment of hsa-miR-15b-5p significantly increased in the anti-pan-Ago RIP complex.

Normalization and Analysis Methods

During the qRT-PCR, cDNA is quantified during the exponential doubling phase. Fluorescence is measured to calculate threshold cycle (Ct) values which quantifies PCR products amplified at a given point in the reaction. The more cDNA templates used to initiate the reaction, the fewer numbers of cycles needed to reach a given threshold.

There are two methods used for qPCR quantitation. The most common method for relative quantitation is the 2^-ΔΔCt method that calculates the relative fold gene expression of samples when performing real-time polymerase chain reactions (31) and standard curve methods (32) that measures absolute numbers of transcripts relative to a standard curve. Since there are no significant differences between the 2^-ΔΔCt and the standard curve methods, it is not necessary to analyze the data with both methods (33). By far, most analyses use relative quantitation since it is easier to perform and is useful to researchers comparing samples under different conditions. For absolute quantitation, an RNA standard curve of the gene of interest is required to calculate the number of copies. In this method, a serial dilution of a known amount (number of copies) of pure RNAs are amplified using the same qPCR program to generate a standard curve. Similar to a protein assay, the unknown signal is compared with the curve to calculate the starting concentration of samples. Proceed to step 12 for the 2^-ΔΔCt method or step 13 for the standard curve method.

• 12The 2^-ΔΔCt method.

  This method uses raw qRT-PCR Ct values to calculate fold changes based on the comparisons between a miRNA of interest and a normalizing miRNA (snU6) as described below.

  • ΔCt of control= (Average Ct of control sample-Average Ct of snU6). The control sample is the control treatment or control time point, etc.

  • ΔΔCt = (Average Ct of sample-Average Ct of SnU6)- ΔCt of control Fold change = 2^-ΔΔCt

• 13Standard curve method.

  Values for miRNA quantities are generated by serial diluting a given cDNA sample produced under the same RT conditions to generate a standard curve. Serial dilutions of samples are then run at the same time, and fictitious values are calculated based on the standard curve.

  Fictitious value = Average of sample/Average of snU6 (or other controls)

  Fictitious values can be determined for each point of a serial dilution of the cDNA to generate a standard curve, so the relative values for an unknown sample can be calculated by comparing its signal to the standard curve (34).

Basic Protocol 3. Primary miRNAs (Pri-miRNA), pre-miRNA and mature miRNA analysis using digoxigenin labeled RNA probes

The most common method for detection of specific miRNAs is Northern blot analysis using radioisotopes; however, this method has some disadvantages of degradation during the assay, low resolution, and the complexities of handling radioisotopes. Therefore, several methods have been developed for non- radioactive labeling and detection of specific nucleic acids. A well-accepted non- radioactive detection of microRNA method has been developed as an alternative method for labeling nucleic acid probes with the cardenolide digoxigenin (DIG) (35). To detect the DIG-labeled RNA-DNA hybrids, a highly specific polyclonal sheep antibody conjugated with alkaline phosphatase are used(35). It has been shown that a 3’-DIG-labeled RNA oligonucleotide probe for miRNA detection is equally sensitive to the standard ³²P-labeled probe (36) following northern blot analysis. In addition, DIG-labeled probes can be detected by a chemiluminescent kit which produces a bright signal in a short time period.

Materials

Ultrapure water

5’ and 3’-DIG-labeled RNA oligonucleotides are synthesized by Proligo Primer & Probes and HPLC purified.

• miR-15b, 5′-TGTAAACCATGATGTGCTGCTA-3′ (5′-DIG and 3′-DIG)

• Scramble-miR used for negative control, 5′- GTGTAACACGTCTATACGCCCA-3′ (5′-DIG)

• U6 snRNA for positive control, 5′-CACGAATTTGCGTG TCATCCTT-3′ (5′-DIG)

UltraPure^™ Agarose (ThermoFisher Cat. 16500500)

NorthernMax^™ 10x Running buffer (MOPS-based buffer containing sodium acetate and EDTA) (Ambion Cat. AM8671)

NorthernMax^™ 1x Running buffer (1:10 dilution from 10x Running buffer with Ultrapure water)

37% formaldehyde (Sigma-Aldrich, Cat. 252549–500ML)

6X glycerol loading buffer (Thermp-Fisher, Cat. R0611)

Thermo Scientific^™ Owl^™ D2 Wide-Gel Electrophoresis System (Cat. 09–528-165)

Ultrapure ethidium bromide 10 mg/ml, ThermoFisher, Cat.# 15585011

0.5µg/ml of ethidium bromide diluted from 10mg/ml ethidium bromide with 1x running buffer

Blot membrane (Bio-rad Zeta Probe GT)

20X Saline-sodium citrate (SSC) buffer (see Reagents and Solutions).

10x SSC buffer (1:1 dilution from 20x SSC with Ultrapure water)

ULTRAhyb-Ultrasensitive Hybridizaiton Buffer (Ambion AM8670)

Whatman 3MM paper (Sigma-Aldrich, Cat. WHA3030861)

Paper towels

A light weight such as a glass plate

A glass tray used for setting up northern blotting experiment

DIG Luminescent detection kit (Roche, 11363514910)

• DIG-labeled control DNA 5µg/ml

• DNA dilution buffer

• Anti-DIG, alkaline phosphatase conjugated antibody

• Blocking Reagent (10% w/v)

• CSPD 11.6mg/ml

Maleic acid buffer (see Reagents and Solutions)

10x Blocking stock solution (dissolve blocking reagent 10% (w/v) in Maleic acid buffer under constantly stirring on a heating block (65°C) or heat in a microwave oven.

1x DIG labeling washing buffer (see Reagents and Solutions)

1x Blocking solution (dilute the 10x blocking solution 1:10 in Maleic acid buffer 0,3%(v/v) Tween 20

1x Detection buffer (see Reagents and Solutions)

1x CSPD working solution (Dilute CSPD(11.6mg/ml) 1:100 in 1x detection buffer)

1x Antibody solution (Dilute Anti-DIG, alkaline phosphatase conjugated antibody 1:10,000, 75mU/ml in 1x blocking solution)

RNA ladder (ThermoFisher Scientific, AM7778)

Kodak BioMax MS film (Sigma, Z363030)

1. Prepare a 1% agarose gel with NorthernMax^™ 1x Running buffer

2. and prepare total RNA as described in basic protocol 1, steps 1–18.

3. Mix 10 μg of total RNA in a solution containing 10 μl ultrapure water, 15 μl of 1x Running Buffer and 5 μl of 37% formaldehyde. Heat samples at 70°C for 10 min in a thermocycler and then place them on ice for 1 min.

4. Add 6 μl of RNA ladder and 6 μl of 6x glycerol loading buffer to each sample from step 2 before loading. Run the gel at 80 to 125 V for at least 2 h.

5. Confirm even loading and the presence of non-degraded RNA in the samples by visualizing the rRNA bands with 20ml of 0.5µg/ml of ethidium bromide in 1x running buffer before performing northern blotting as described in basic protocol 1 (steps 53).

6. Rinse the gel gently with water and cut the blot membrane to the same size as the gel. Equilibrate the membrane in RNAase free water for 10 minutes followed by in 10x SSC.

7. Assemble the transferring setup in a glass tray containing 200 ml of 10x SSC. Sheets of Whatman 3MM paper are first moistened with 10x SSC and are then layered with their ends dipped in 10x SSC within a glass tray to act as wicks. Gel, membrane, three pieces of Whatman 3MM paper moistened with 10x SSC and cut to the size of the membrane are stacked on top of each other, respectively. Then place a stack of paper towels 5–10 cm high and cut to the size of the gel on top. A light weight such as a glass plate is then evenly stacked on top to facilitate contact between the stacked layers.

8. Allow the transfer to occur at room temperature for at least 6 hours or overnight.

   Northern blots are prehybridized at 65°C for 1 hour using Ultrahyb-Ultrasensitive Hybridizaiton Buffer and subjected to hybridization with 3’-DIG-labeled RNA probe (100ng/ml) for the specific miRNA such as hsa-mir-15b together with DIG- abeled U6 probe overnight at room temperature (37).

9. Use the DIG Luminescent detection kit to detect the DIG labeled probe. Blots were incubated in 1x blocking solution for 30 min and then in 1x antibody solution for 30 min. After washing twice in washing buffer and equilibration in detection buffer, blots are incubated with 1x chemiluminescent substrate CSPD and exposed to Kodak Biomax film.

Basic Protocol 4. Mapping of primary miRNA and Drosha cleavage products by Rapid amplification of cDNA ends (RACE)

Primary miRNAs are transcribed from genome DNAs in the nucleus. They can be transcribed as noncoding long RNAs, or can be part of introns which are excised out of primary gene-coding transcripts during splicing (38). It is important to identify primary miRNA transcripts, because it allows the characterization of the mature miRNA biogenesis process and to identify key factors in this process. RACE (Rapid Amplification of cDNA Ends) methods have been used for the identification of 5’ and 3’ primary miRNA transcript ends (39, 40).

This method can also be used to identify and characterize cleavage sites processed by Drosha (40, 41).

Material

GeneRacer Kit (Invitrogen, L150201)

• DEPC water

• RNaseOut (40U/ μl) (manufacturer/product # ?)

• Calf Intestinal Phosphatase (CIP 10U/ μl)

• 10x CIP buffer (in the kit)

• Tobacco Acid Pyrophosphatase (TAP 0.5U/ μl)

• 10x TAP buffer (in the kit)

• T4 RNA Ligase

• 10x T4 RNA Ligase Buffer

• 10 mM ATP

• Phenol:Chloroform (Phenol:chloroform:isoamyl alcohol 25:24:1)

• Mussel Glycogen (10mg/ml) in DEPC water

• 3M Sodium Acetate pH5.2 in DEPC water

• GeneRacer 5’Primer (10 µM in DEPC water, 71.5 ng/µl)

  5′-CGACTGGAGCACGAGGACACTGA-3′

• GeneRacer 5’Nested Primer (10 µM in DEPC water, 81.3 ng/µl)

  5′-GGACACTGACATGGACTGAAGGAGTA-3′

• GeneRacer 3’Primer (10 µM in DEPC water, 76.9 ng/µl)

  5′-GCTGTCAACGATACGCTACGTAACG-3′

• GeneRacer 3’Nested Primer (10 µM in DEPC water, 71.1 ng/µl)

  5′-CGCTACGTAACGGCATGACAGTG-3′

• SuperScript III Reverse Transcriptase (RT) 200U/µl

• 5x First Strand Buffer

• 0.1 M DTT

• RNaseH (2U/ μl)

• Random primers (N₆) (100ng/μl, 54µM))

• Generacer Oligo dT primer (900ng/ml, 50µM))

• dNTP mix (10 mM each)

High Fidelity Taq (Invitrogen, Cat.# 11304011)

1.5 M glycine in DEPC water

Jurkat cell line, Clone E6–1 (ATCC, TIB-152^™)

Hela cell line (ATCC®CCL-2^™)

Fetal Bovine Serum, dialyzed (Gemini, Cat.# 100–108)

Penicillin/Streptomycin (10,000U/ml) (GibcoTM, Cat.# 15140122)

Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC® 30–2002^™) w/10%(v/v) Fetal Bovine Serum (FBS) and 1%(v/v) Penicillin/Streptomycin mix (500 ml DMEM +50 ml FBS +5 ml Pen/Strep)

Cross-Linking and Immuno-Precipitation (CLIP) washing buffer (see Reagents and Solutions)

16% Paraformaldehyde (formaldehyde) aqueous solution 10 × 10 ml. (Electron Microscopy Science, CAS#30525–89-4)

0.1% Formaldehyde solution diluted from 16% paraformaldehyde solution with DEPC water

Protein A Sepharose beads (GE healthcare, 17–5138-01)

PK buffer (see Reagents and Solutions)

PK-7M Urea buffer (see Reagents and Solutions)

10 µM dNTP (deoxyribonucleotide triphosphate)

QIAquick PCR purification kit (Qiagen, Cat.# 28106)

Phusion High-Fidelity (HF) DNA polymerase (Thermo Fisher Scientific, F-530L)

Bioruptor II Type 6 (COSMO BIO, Cat.# TOS-BR2006A)

Troemner^™ Talboys^™ Advanced Digital 1000MP Microplate Shaker (Troemner^™ 980178, Cat.# 02–217-993)

DNaseI (Takara, Cat.# 2270B)

Alkaline phosphatase (Takara, Cat.# 2120B)

T4 polynucleotide kinase (PNK, Takara, M0201S)

Protein A Sepharose beads (GE healthcare, 17–5138-01)

Rabbit monoclonal DROSHA antibody (Cell Signaling, #3364)

10% Novex^™ TBE-Urea Gels (ThermoFisher Scientific, EC68755BOX)

2x Novex^™ TBE-Urea Sample Buffer (Invitrogen^™ Cat.# LC6876)

Stacked tubes (Made by puncturing the bottom of a sterile, Rnease-free, 0.5 ml microcentrifuge 4–5 times with a 21-gauge needle and placing it into a sterile, round-bottom, nuclease-free, 2 ml microtube)

Identify the ends of Primary Transcripts by 5’ and 3’ RACE.

This experiment requires 1–5 μg of total RNA.

• 1Conduct basic protocol 1 steps 1–18 on 10⁶ Jurkat T cells to get the total RNA.
• 2Dephosphorylate RNA by incubating total RNA in 7 μl DEPC H₂O (isolated from basic protocol 1) with 1 μl of RNaseOut (40U/ μl) and 1 μl of calf intestinal phosphatase (CIP 10U/ μl) and 1 μl of 1x CIP buffer (Invitrogen) in a 10 μl of reaction at 50°C for 1h.
• 3After incubation, centrifuge the tube at 10,000 rpm for 30 seconds and place on ice.
• 4For RNA purification, add 90μl of DEPC-treated water, 100μl acid phenol:chloroform. Vortex thoroughly. Centrifuge at room temperature at 15,000 rpm for 5 minutes.
• 5Transfer the aqueous layer to a new tube (around 100μl) and add 2μl 10 mg/ml mussel glycogen in DEPC water, 10 μl 3M sodium acetate in DEPC water, pH5.2, and mix well. Add 220μl 95% ethanol and vortex briefly.
• 6Freeze the samples on dry ice for 10 minutes before proceeding to the next step or storing at −20°C overnight. (Do not store the RNA in DEPC-treated water. Store RNA in ethanol at −20°C.)
• 7Centrifuge at 15,000 rpm for 20 minutes at 4°C. Identify the position of the pellet and remove the supernatant by pipet. Be careful not to disturb the pellet.
• 8Rinse pellet with 0.5 mL cold 70% ethanol, invert several times, and vortex briefly. Centrifuge 5 minutes at 15,000 rpm at 4°C. Remove ethanol carefully and discard it.
• 9Centrifuge again to collect remaining ethanol.
• 10Carefully remove the remaining ethanol by pipet and air-dry the pellet for 2 minutes at room temperature.
• 11Resuspend the RNA pellet in 7μl of DEPC-treated water. Decap the RNA by adding 1μl of RNaseOut and 1 μl of tobacco acid pyrophosphatase (TAP) to the RNA in 1μl of 1x TAP buffer. Incubate at 37°C for 1h and extract RNA as described step 3 to 9. Resuspend the RNA pellet in 7μl DEPC-treated water.
• 12Incubate 1 tube containing the pre-aliquoted, lyophilized GeneRacer RNA Oligo (0.25 µg) with the 7 μl of dephosphorylated, decapped RNA at 65°C for 5 min and place it on ice for 2 min followed by addition of 1 μl of RNaseOut, 1 μl of T4 RNA ligase, 1 μl of 10mM ATP, and 1 μl of 10x T4 ligase buffer. Incubate at 37°C for 1 h. After incubation, purify the RNA as described from step 3–9. Resuspend the pellet in 10 μl DEPC-treated water.
• 13Prepare a mixture of 1μl of 50 μM GeneRacer oligo dT primer, 1μl of 10 μM dNTP (deoxyribonucleotide triphosphate), and 1μl of DEPC-treated water and add to the ligated RNA (from basic protocol 4 step 4) to begin cDNA synthesis. Incubate the solution at 65°C for 5 min and 4°C for 1 min.
• 14Subsequently add a premixed solution containing 4 μl of 5x First Strand Buffer, 1 μl of 0.1 M DTT, 1μl of RNaseOut, and 1μl of SuperScript III reverse transcriptase. Incubate at 50°C for 1 h, 70°C for 15 min, and ice for 2 min. Then, add 1μl (2U/ μl) of RNase H and incubate at 37°C for 2 min.
• 15To characterize the 5’ end of the cDNA, perform the PCR reaction with the GeneRacer 5’ Primer and a gene-specific reverse primer 3’ to the mature miRNA using a High Fidelity Taq polymerase. (Purify the PCR reaction using a PCR purification system and elute in 50 μl of DEPC H₂O.
• 16Use 1 μl of the first PCR reaction (step 15) as a template to perform nested RACE by adding a second set of primers corresponding to the GeneRacer 5’ nested oligo (downstream of the original forward primer) and a gene-specific primer (upstream of the original reverse primer). Set up PCR conditions with 25–30 cycles of amplification in a 50 μl reaction using High Fidelity Taq polymerase.
• 17Analyze PCR products in an agarose gel.

The specific band corresponding to the gene of interest should be verified by size, diagnostic restriction digestions or gel purification before proceeding to cloning into a TOPO vector. The cloned DNA can be transformed into bacteria for plasmid replication and purification. Purified plasmid is sequenced to identify the primary miRNA 5’ end(s).

Similar methods can be used as described above to characterize the 3’ end of the cDNA by using the GeneRacer 3’primer and a 3’ nested primer with a 5’gene-specific primer and a nested primer corresponding to the mature miRNA (see Gene racer kit instruction manual).

Map Drosha cleavage sites in primary miRNA by DROSHA fCLIP-seq (formaldehyde crosslinking immunoprecipitation and sequencing).

• 18Grow Hela cells in DMEM w/ 10% FBS and 1% Pen-Strep in a T75 flask at a horizontal position to 5 × 10⁶ cells per flask. After rinsing with PBS, the adherent cells are fixed with 18 mL of PBS-containing 0.1% formaldehyde solution in the flask at RT for 10 min.
• 19Add 2 mL of 1.5 M glycine in DEPC water to the flask at RT for 10 min to quench formaldehyde crosslinking.
• 20Collect the 20 ml of fixed cells and resuspend in 550 μl of Cross-Linking and Immunoprecipitation (CLIP) wash buffer for 10 min to lyse the cells (42).
• 21The cell lysate is then sonicated by using bioruptor II and treated with 20 μl of DNaseI at 37°C for 10 min. The lysate is then centrifuged at 16,100g for 10 min at 4°C, and the supernatant (about 20 mls) is utilized for immunoprecipitation.
• 2225 μl of protein A Sepharose beads is prewashed three times with 1 mL of CLIP wash buffer in a microcentrifuge tube using a low speed (1000–2000RPM) for 3 minutes. Add 20 μg of rabbit monoclonal DROSHA antibody to the beads in 400 μl of CLIP wash buffer for 4 h at 4 °C. The antibody-bound beads are then washed twice with CLIP wash buffer and incubated with the prepared lysate supernatant (step 21) at 4°C for 4h.
• 23After incubation, wash the immune-precipitates six times with CLIP wash buffer as described in step 22 and discard the supernatant. Elute the crosslinked DROSHA-RNA complexes from beads by incubating the immunoprecipitants in 300 μl of PK-7M Urea buffer at 25°C for 2 hr on the Troemner shaker at 950 rpm.
• 24Remove ~300 μl of supernatant after a brief centrifugation at 1500 rpm for 2 minutes.
• 25The crosslinked RNAs (step 24) are then separated from DROSHA proteins by addition of 300 μl of 20 mg/ml Proteinase K to the supernatant and incubating the solution at 65°C for 12 h at 1000 rpm.
• 26The RNAs are then isolated from solution by acid phenol:chloroform extraction as described in this protocol step 3 to 10. About 500–1000 ng of RNA is usually obtained at this step.
• 27Purified RNAs are sequentially treated with 1U of DNaseI, 0.1U alkaline phosphatase, and 10U of T4 polynucleotide kinase (PNK) at 37°C for 1 hour. RNA products are isolated from each treatment solution by acid phenol:chloroform extraction as described in this protocol step 3 to 10.
• 28After PNK treatment, RNAs are run on a 10% acrylamide Urea-PAGE gel with 1x TBE buffer and then the 25–160 nt RNA fragments are cut and transferred into a 0.5 ml microcentrifuge stacked tube. RNAs are purified from the excised gel (steps 26–31, basic protocol 1) and are ligated to 3’ adapters by using T4 RNA ligase as described in basic protocol 1 step 35 to 38.
• 29Ligation products are electrophoresed using a 10% acrylamide Urea-PAGE gel and purified from the gel (basic protocol 1, steps 26–31).
• 30The 3’ adaptor-ligated RNAs are ligated to 5’ adapters (as described in basic protocol 1 step 31 to 34 by T4 ligase 1 ) and reverse transcribed by using SuperScript III Reverse Transcriptase ) and RT primers as described in basic protocol 1 step 40–46.
• 31The RNA is then reversed transcribed into cDNA and amplified by PCR utilizing Phusion HF polymerase, 5’ PCR primer and 3’ PCR primers as described in basic protocol 1 step 47–48.
• 32PCR products are purified using 6% acrylamide gels and sequenced utilizing an Illumine HiSeq 2500 platform as described in the basic protocol 1 step 48–63.

An isotype negative control antibody (rabbit IgG) to assess non-specific binding should be utilized, since background binding is usually the result of immunoglobulin binding non-specifically to other proteins or RNAs. Fold-enrichment of the specific antibody-coupled protein should be calculated in comparison to that from the isotype control samples.

Basic Protocol 5. Electrophoretic Mobility Shift Assays (EMSA) of precipitated miRNPs

EMSA is a widely used methodology to identify large ribonucleoprotein complexes (Pham JW 2004) or the proteins which bind to a radiolabeled miRNA. A simple EMSA experiment can be performed with radiolabeled miRNAs and crude extracts as demonstrated here utilizing the miRNA has-mir-15b from Hela cells(43). It is important to handle the radioactive materials safely. In addition to wearing personal protective clothing, the radioactive materials work area should also be set up with absorbent paper and dedicated equipment, which are only used for radioactive material work. All persons should have radiation safety training before handling radioactive material and ensure radioactive waste is properly labeled and placed in a leak-proof radioactive waste container free of external contamination.

Materials:

Jurkat cell line, Clone E6–1 (ATCC, TIB-152^™)

Dynabeads^™ protein A for immunoprecipitation (Invitrogen, Cat.#10001D)

RPMI-1640 medium (ATCC® 30–2001^™)

Fetal Bovine Serum, dialyzed (Gemini, Cat.# 100–108)

Penicillin/Streptomycin (10,000U/ml) (GibcoTM, Cat.# 15140122)

High Fidelity Taq (Invitrogen, Cat.# 11304011)

T4 RNA ligase 1(ssRNA ligase) (New England Biolabs, Cat.# M0204S)

T4 RNA ligase 2 (dsRNA ligase) (New England Biolabs, Cat.#M0239S)

Phusion High-Fidelity (HF) DNA polymerase (Thermo Fisher Scientific, F-530L)

Cell extraction buffer (Invitrogen, Cat.# FNN0011) or NP40 cell lysis buffer (Invitrogen, Cat.# FNN0021)

salmon-sperm DNA(ssDNA), sheared (10mg/ml) (Invitrogen, Cat.# AM9680)

Bio-Rad protein assay dye reagent concentrate, 450ml (Bio-rad, #5000006)

RIP WB buffer (0.1M NaPO4, 0.1%Tween 20, pH 8.2)

Bis(sulfosuccinimidyl) substrate (BS³) BS conjugation buffer (5mM BS³ in conjugation buffer containing 0.5μg/μl ssDNA)

Benchmark Roto-Therm^™ Incubated Tube Rotators (The Lab Depot, Inc. Cat.#H2024))

RIP High salt wash buffer (see Reagents and Solutions)

RIP PK buffer (see Reagents and Solutions)

PK-7M Urea buffer (see Reagents and Solutions)

Proteinase K solution (5 mg of proteinase K in 1 mL of PK buffer)

Trizol RNA Isolation Reagents (Invitrogen Cat.# 15596026)

miRNA binding buffer (see Reagents and Solutions)

6x native gel loading dye (New England Biolabs Inc., Cat.# B7021S)

Native gel (16cm x 16 cm gel of 0.8 mm thickness with 1xTBE and 3–12%, ThermoFisher Scientific, BN1001BOX)

BioRad Model 583 Gel Dryer (BioRad, Cat.# 1651745)

Kodak®BioMax® MS film (Sigma-Aldrich, Cat.# Z363030–50EA)

RNase A 50U/mg (Sigma-Aldrich, Cat.# 10109142001)

RNase A buffer (see Reagents and Solutions)

XT sample buffer 4x (Bio-Rad #1610791)

Gamma-³²P ATP (6000 Ci/mmol, PerkinElmer, BLU002Z250UC)

Synthetic miRNA (50 pmol, Dharmacon, Inc.(44))

RIPAb+TM pan Ago mouse monoclonal antibody/Primer Set (Millipore, Cat.# 03–248)

• Anti-pan Ago (Mouse Monoclonal), Part # CS207301

• Normal Mouse IgG, Part # CS200621

RNA immunoprecipitation (RIP)

• 1Dynabeads protein A can be utilized for this procedure. 5×10^6 Jurkat T cells cultured in RPMI-1640 medium with 10% Fetal Bovine Serum (FBS) are harvested and lysed using 200 μl of cell extraction buffer or NP40 cell lysis buffer for 10 minutes at room temperature.
• 2Samples are then centrifuged at 16,000g in a microcentrifuge tube at 4°C for 15 min. Transfer the supernatants to a new microcentrifuge tube and determine the protein concentration of protein by a Braford protein assay (about 150~200 µg total protein generally obtained).

  A minimum of 50 μg of lysate protein is required for RIP for each sample. Similar quantities of protein should be utilized for each condition.

• 3Wash 50 μl of untreated Dynabeads with 100 μl of WB buffer, spin down at 1500 rpm, and remove the supernatant. Repeat the washing step a second time. Add lysate from step 2 to dynabeads to preclear lysate. Rotate at 4°C for 1h. Spin down the beads at 1500 rpm for 3 minutes and then transfer the pre-cleared lysate to a new tube on ice.
• 4Wash 50μl of fresh Dynabeads per sample with 100 μl of WB buffer as describe in step 3. Block beads with 180 μl of WB buffer and 20 μl of 10 μg/μl sheared, salmon-sperm DNA (ssDNA) to reduce background binding for 30 minutes on a tube rotator.
• 5Add 10 μg of anti-pan Ago antibody to the beads (use same amount of normal mouse IgG as a control) and incubate on rotator at room temperature for 10 min. Wash the antibody-bead complex twice with 100 μl BS conjugation buffer by spinning at 1500 rpm for 3 minutes. Incubate beads in 250 μl BS conjugation buffer at RT for 30 min with rotation. The cross-linking reaction is subsequently quenched by addition of 12.5 μl of 1 M Tris-Cl pH 7.4 and is incubated at RT for an additional 15 min with rotation.
• 6Spin the beads down at 1500 rpm for 3 minutes and then add 100 μl of NP40 cell lysis buffer. Repeat this wash a second time. Then add precleared lysate from the previous step 3 to these beads. Rotate at 4°C overnight.
• 7Spin the beads down for 3min at 1500 rpm, and add, while on ice, 100 μl of RIP wash buffer for 1 minute. Repeat this wash a second time and then repeat the rinse using the same volume of RIP high salt wash buffer and RIP PK buffer once sequentially.
• 8Resuspend beads in 100 μl of RIP PK buffer and remove 15 μl of the mixture for an IP efficiency analysis by western blotting. Remove excess RIP PK buffer by spinning the sample down at 1500 rpm for 3 minutes and incubate beads in 100 μl of proteinase K solution (5mg/ml) at 37°C for 20 min in a Thermomixture R set at 1200 rpm.
• 9Quench the reaction by adding 100 μl of PK-7M Urea buffer and incubate the sample at 37°C for 20 min at 1000 rpm.
• 10Total and immunoprecipitated RNA can be extracted by adding acid phenol: chloroform directly to an aliquot of input protein lysate or the proteinase K treated solution as described in this Protocol step. Resuspend purified RNA samples in 10 μl of DEPC-treated H₂O. Quantity and quality of the RNA can be measured using a nanodrop. 5 ng −1 ug of RNA is usually utilized for the qRT-PCR analysis in the next step. (Isotype control IgG was used to normalize the hsa-mir-15b that is pulled down by argonaut complexes. (Fig.1b and 1c)
• 11Purified RNA can be used for small RNA-seq or RACE labeling, and qRT-PCR as describe in the protocol 4 to map the Drosha cleavage sites or quantitate the expression of mature miRNA, pri-miRNA or pre-miRNA as described in the basic protocol 3.

Electrophoretic Mobility Shift Assays (EMSA) of precipitated miRNPs

• 12To radiolabel synthetic miRNA (synthesized miRNA are obtained from Dharmacon Inc.), mix 1 μl of 50 μM synthetic miRNA (50 pmol of hsa-mir-15b) as described in the previous studies(43)), 5 μl of gamma-³²P ATP (6000 Ci/mmol, 2.5 μl of 10x T4 PNK buffer, 2.5 μl of T4 polynucleotide kinase, and 14 μl of RNase-free water (45).
• 13The mixture is incubated at 37°C for 30 min followed by elimination of the free gamma-³²P ATP by passing the sample through a Sephadex G-25 column.
• 14Prepare a total of 10 μl of miRNA binding reactions by mixing 5×10^5 c.p.m radiolabeled miRNA and Jurkat cell extract (as described in step 1) in the miRNA binding buffer. The mixture is then incubated at RT for 45 min followed by adding 2 ul of 6x native gel loading dye before loading to the native gel.
• 15After loading samples, run the native gel at 100 V at 4°C until the bromophenol blue (BPB) dye migrates within 3 cm of the bottom of the gel. Remove the gel and place onto Whatman 3MM paper and dry it using a gel dryer. Detect signals by autoradiography or phosphoimaging.
• 16Alternatively, to recover the RNA from the gel, 150 μl of miRNA binding reaction can be separated on a nondenaturing gel. Wrap wet gel in a transparent plastic film and expose it to a BioMax MS film for 1 h. After autoradiography, cut the gel bands that contain radioactive signals corresponding to miRNPs of interest.
• 17Remove the gel slice on a clean glass plate on ice and expose them to 254 nm UV light for 30 seconds using a Stratalinker UV Crosslinker.

  Cross-linking of radio-labeled miRNA with miRNA binding proteins allows the transfer of the radioactive label to miRNA binding proteins and can be visualized on SDS-PAGE.

• 18Gel slices are then minced into small pieces using a razor blade and incubated with 200 μl RNase A buffer (250 U/mL) at RT for 30 min.
• 19After adding 50μl of XT sample buffer 4x (Bio-Rad) and shaking on a vortex mixer for 3 h, proteins are analyzed by an SDS- PAGE 4–12% gradient gel followed by autoradiography. At this step, you should be able to observe the proteins bands binding to the miRNA.
• 20To eliminate signal that is not produced by miRNA complexes, 20 μl of miRNA binding mixture is carried through steps 17–19 above.
• 21This data from this gel should reveal the proteins that binds to the miRNA.

Alternate Protocol 1. miRNP purification by Biotinylated 2’-O-methyl Oligonucleotides

Complementary 2’-O-methyl oligonucleotides block miRNA function in vitro and in vivo (46). They can also capture proteins binding to miRNA in miRNP complexes by using Biotinylated 2’-O-methyl Oligonucleotides, which allows identification of proteins in the complex (47).

Materials

Streptavidin paramagnetic beads (Dynabeads, MyOne Streptavidin T1 10 mg/ml; Invitrogen)

miRNP Washing buffer (see Reagents and Solutions)

miRNP Washing buffer containing 2 mM DTT (see Reagents and Solutions)

miRNP Washing buffer containing 2 mM DTT and 0.01% (v/v) Triton X-100 (see Reagents and Solutions)

miRNP Washing buffer containing 2 M potassium acetate, 2 mM DTT, and 0.01% Triton X-100 (see Reagents and Solutions)

XT sample buffer 4x (Bio-Rad #1610791)

XT reducing agent (20x) (Bio-Rad #1610792)

Criterion XT Bis-Tris 4–12% gradient gel (Bio-Rad)

XCell Sure Lock Mini-Cell electrophoresis unit (Invitrogen, Part#E10001)

Electrophoresis power supply

Protocol Steps:

1. 100 μl of Streptavidin paramagnetic beads are washed and incubated with 5’ biotinylated, 2’-O-methyl complementary (capture) oligonucleotides (500 pmol) according to the manufacturer’s instructions. For example, the capture oligonucleotide (entirely 2’-O-methyl ribose) were 5’-biotin-UCUUCACUAUACAACCUACUACCUCAACCUU-3’, which is fully complementary to let-7) (47). It is retained on ice after resuspension in 100 μl of washing buffer containing 2 mM dithiothreitol (DTT).

2. Replace the buffer with 100 μl of cell extract (as described in basic protocol 5, step 1). Incubate at RT for 30 min with gentle rotation.

3. Remove the supernatant and wash the beads five times with washing buffer containing 2 mM DTT and 0.01% (v/v) Triton X-100, followed by five washes with washing buffer containing 2 M potassium acetate, 2 mM DTT, and 0.01% Triton X-100.

4. Resuspend the beads in a final volume of 28 μl of water and add 10 μl of XT sample buffer (4x) and 2 μl XT reducing agent (20x). Heat at 95°C for 5 minutes

5. Subject the sample to denaturing gel electrophoresis using a Criterion XT Bis-Tris 4–12% gradient gel. proteins can be analyzed by western blotting with antibodies against target proteins.

6. RNAs within bead-complexes or in the supernatant can be extracted by acid phenol: chloroform and analyzed by qRT-PCR or northern blotting as described in the basic protocol 2 and 3.

Basic Protocol 6. Controlling miRNA expression in Jurkat cells by miRNA mimics and inhibitors transfection, to enable microRNA functional analyses

This method can be used to confirm expression changes in miRNA targets by its associated miRNA. After identifying new miRNAs and their potential targets, miRNA mimics or inhibitors are used to confirm experimentally that a miRNA is involved in the regulation of a specific gene of interest or a specific pathway of interest (48, 49). Unlike siRNA mediated gene silencing in which complete sequence complementary is required in most cases, miRNA-mediated regulation can be achieved by partial complementariness between miRNA and mRNA with as few as six to seven complementary nucleotides near the 5’-end of the miRNA (the seed region). siRNAs are highly specific with only one mRNA target; however, miRNAs have multiple targets. Each target gene can also be regulated by multiple miRNAs.

miRNA mimics or inhibitors are powerful tools in analyzing miRNA functions, since they result in overexpression or knockdown effects of endogenous miRNA respectively, which allow easier observation of the miRNA effects on target gene expression levels.

miRIDIAN mimics and hairpin inhibitors from Dharmacon have been used by many laboratories (Dharmacon.horizondiscover.com).

There are two important variables for the mimics or inhibitors to function well in the cells - their concentrations and the cell types used for the experiments, according to the user guide by Dharmacon. In our hands we have used them in both Jurkat cell lines and human isolated PBMCs.

Materials

MiRIDIAN mimics or hairpin inhibitors, MiRIDIAN mimic negative controls and hairpin inhibitor negative controls (Dharmacon)

Jurkat cell line, Clone E6–1 (ATCC, TIB-152^™)

Fetal Bovine Serum, dialyzed (Gemini, Cat.# 100–108)

Penicillin/Streptomycin (10,000U/ml) (GibcoTM, Cat.# 15140122)

RPMI-1640 medium (ATCC® 30–2001^™)

PBMC isolated from human healthy donor (NIH blood bank)

Lonza Nucleofector^™ 2b (Lonza, Cat.#AAB-1001)

Amaxa Cell Line Nucleofector kit V (Lonza, Cat#, vvca1003)

miRNA mimics and inhibitors and negative controls (Dharmacon)

• miRIDIAN microRNA mimic hsa-miR-15b-5p, (Dharmacon, C-300587–05-0020 20nmol)

• miRIDIAN mimic Negative Control, miRIDIAN microRNA Mimic Negative Control#1 (Dharmacon, CN-001000–01-05 5nmol)

• miRIDIAN microRNA Hairpin Inhibitor, hsa-miR-15b-5p, (Dharmacon, IH-300587–07-0020 20 nmol)

• miRIDIAN microRNA Hairpin Inhibitor Negative Control #1, (Dharmacon, IN-001005–01-05 5nmol)

Accell siRNA delivery media (Dharmacon, cat# B-005000–500)

1. 5× 10^6 Jurkat cells in RPMI-1640 with 10% FBS were harvested by centrifugation at 800 rpm for 10 min at room temperature. Cell pellets are washed once with 1xPBS and resuspended in 100μl lonza transfection buffer (supplemented with the cell line nucleofector kit V).

2. Pre-warm 3 mls of RPMI media per well in a 12 well plate at 37°C with 5% CO₂ for 15min.

3. Add 1 μl of mimics or inhibitors (100 μM) into the 100 μl of mixture and transfer the 101 μl mixture into a cuvette to perform the electroporation using a Jurkat cell program on a Lonza Nucleofector^™ 2b device.

4. After each electroporation, add 0.5 ml of pre-warmed media from one well of a 12-well plate into a cuvette to recover cells and transfer cells back to the well and incubate at 37°C with 5% CO₂ for 48 hours.

5. After 48 h incubation, cells are harvested and total RNAs are isolated as described in basic protocol 1. The miRNA expression level and its target mRNA level are then measured by qRT-PCR or northern blotting as described in basic protocol 2, 3 and 4.

Reagents and Solutions

All solutions are made with ultrapure water, and can be stored in the refrigerator at 4°C for up to 1 year unless otherwise noted.

Agrose Running buffer 1x (40mM MOPS, pH 7.0, 2 mM EDTA)

6X glycerol loading buffer (30% glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol)

20X Saline-sodium citrate (SSC) buffer (3M sodium chloride and 300 mM trisodium citrate adjusted to pH 7.0 with HCl).

Maleic acid buffer (0.1M Maleic acid, 0.15 M NaCl; adjust with NaOH (solid) to pH 7.5) (20°C)

1x DIG labeling washing buffer (0.1M Maleic acid, 0.15 M NaCl; pH 7.5) (20°C)

1x Detection buffer (0.1M Tris-HCl, 0.1M NaCl, pH9.5) (20°C)

Cross-Linking and Immuno-Precipitation (CLIP) washing buffer (1X PBS, 0.1% SDS, 0.5% deoxycholate and 0.5% NP-40)

PK buffer (200 mM Tris-HCl pH7.4, 100 mM NaCl, 20 mM EDTA, 2% SDS)

PK-7M Urea buffer (200 mM Tris-HCl pH7.4, 100 mM NaCl, 20 mM EDTA, 2% SDS, and 7 M Urea)

RIP WB buffer (0.1M NaPO4, 0.1%Tween 20, pH 8.2)

RIP conjugation buffer (20 mM NaPO₄, 150 mM NaCl)

Bis(sulfosuccinimidyl) substrate (BS³) conjugation buffer (5mM BS³ in conjugation buffer containing 0.5μg/μl ssDNA)

RIP wash buffer (1x PBS pH 7.4, 0.1%SDS, 0.5% sodium deoxycholate, and 0.5% NP-40).

RIP high salt wash buffer (5x PBS pH 7.4, 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% NP-40)

RIP PK buffer (100 mM Tris-Cl pH7.4, 50 mM NaCl, and 10 mM EDTA)

Proteinase K solution (5 mg of proteinase K in 1 mL of PK buffer)

miRNA binding buffer (20 mM Tris pH 7.5, 1 mM magnesium acetate, 1 mM calcium chloride, 0,01% Nonidet P-40, 2 mM ATP, 250 ng/μl E.coli tRNA)

RNase A buffer (250 U/mL in 50 mM Tris-HCl pH 7.5)

miRNP Washing buffer (30 mM HEPES-KOH at pH 7.4, 100 mM potassium acetate, 2mM magnesium acetate)

miRNP Washing buffer containing 2 mM DTT (30 mM HEPES-KOH at pH 7.4, 100 mM potassium acetate, 2mM magnesium acetate, 2mM DTT)

miRNP Washing buffer containing 2 mM DTT and 0.01% (v/v) Triton X-100 (30 mM HEPES-KOH at pH 7.4, 100 mM potassium acetate, 2mM magnesium acetate, 2mM DTT, 0.01% v/v Triton X-100)

miRNP Washing buffer containing 2 M potassium acetate, 2 mM DTT, and 0.01% Triton X-100 (30 mM HEPES-KOH at pH 7.4, 2M potassium acetate, 2mM magnesium acetate, 2mM DTT, 0.01% v/v Triton X-100)

Commentary

Background information

miRNAs are small, highly conserved RNA molecules that act as key regulators of development, cell proliferation, cell cycle and cell differentiation. More and more evidence also implicates dysregulation of miRNA as being involved in the pathogenesis of human diseases such as cancers, metabolic diseases, neurological disorders, infectious diseases, and other illnesses (50). miRNAs have been shown to be capable of inhibiting the translation and/or stability of target messenger RNAs. Since a miRNA often simultaneously regulates multiple mRNAs of a cellular process, a small change in miRNA expression may have large effects on a given biological process. It is currently thought that around two third of all human genes may be regulated by miRNA molecules (51). Thus, analysis of primary, precursor and mature miRNA levels and characterization of their targets and phenotypic changes is critical to the understanding of certain disease processes.

To measure the different expression levels of miRNAs, many researchers have created global miRNA expression profiles by utilizing small RNA/microRNA sequencing, which requires validation for each individual microRNA expression by using TaqMan MicroRNA assay sets or northern blotting analysis. This approach is also used for detecting the expression level of primary and precursor miRNA by designing specific RT primers.

Detection and quantitation of primary, precursor and mature miRNA expression can also be performed by northern blotting and digoxigenin-labeled probes (non-radioactive probe). To map the Drosha cleavage sites in primary and precursor miRNA, GeneRacer RACE labeling provides a useful tool to label 5’ end and the 3’ end of the cleavage fragments for further qPCR and sequencing.

To investigate the function of proteins involved in protein-microRNA interactions, EMSA and Biotinylated 2’-O-methyl Oligonucleotides targeted miRNP provide useful tools to identify the proteins within miRNP complexes.

For microRNA functional analysis, artificially up-regulating miRNAs by adding miRNA mimics can help identify gain-of-function phenotypes; on the other side, down-regulation or inhibition of miRNA experiments can identify loss-of-function phenotypes. The combination of both can also be used for screening genes and cellular biological processes regulated by specific miRNAs. Once a gene is identified as a target of a specific miRNA, a further investigation of miRNA function will include miRNA-target interaction and the impact on the target gene’s mRNA and protein expression.

Deep sequencing allows the discovery of novel miRNAs. For analysis, there are several software tools publicly available based on different algorithms. miRDeep is one of the most commonly used software to search for novel miRNAs (52).

An overview of online resources of miRNA sequences, expression and target predictions is summarized in a review (53).

Taken together, these studies may provide effective methods to study the role of pharmacological intervention within these orchestrated cellular programs. The powerful tools described in this review thus provide fundamental approaches for analyzing the function of miRNA both in vitro and in vivo that could profoundly shape the future of drug development and therapy for human diseases.

Critical Parameters and Troubleshooting

In basic protocol 1, to perform small RNA sequencing, sufficient amounts of total RNAs (at least 10 μg) are required for sequencing. Lower amounts may result in inefficient ligation and low yield. It is also important that the input RNAs have in sufficient quality; otherwise, it may result in low yield or failure of RNA isolation. The RNA integrity can be assessed by an Agilent Technologies 2100 Bioanalyzer and the RNA Integrity number (RIN) value should be greater than 8.

When recovering small RNAs from gels, it is important to excise a gel region corresponding to the correct range based on the size of a standard RNA ladder. In the basic protocol 4, the steps of DNAse I digestion are important for eliminating DNA contamination for the miRNA qRT-PCR and northern blotting analysis. In RIP protocols, the preclearing of the cell lysate is a critical step that allows the reduction of non-specific binding. A negative isotype control (IgG) should also be included in experiments to distinguish specific binding from non-specific binding. Finally, in the application of miRNA mimics or inhibitors in vivo, it is important to use negative controls of mimics and inhibitors for further comparison. Their working efficiency is dependent on the concentrations and cell types.

Trouble Shooting

In the basic protocol 2, 1–10 ng are recommended for a typical qRT-PCR. However, for low abundancy genes, more input RNA is required. One way is to titrate the amount of input, up to 1μg of total RNA. If this is not sufficient, doubling the amount of RT enzyme may improve yields. qRT-PCR can be utilized not only for the calculation of fold changes, but also can be used for absolute quantitation of the exact number of expressed copies. For that purpose, a synthetic microRNA is required to create a standard curve.

Primer design and selection for pri-miRNA and precursor miRNA in qRT-PCR. Primary miRNA can be amplified by selecting primers near to but not within the pre-miRNA hairpin. In contrast, precursor miRNAs can be amplified by designing primers included in the hairpin. Because all precursor miRNA primers are also present within their respective pri-miRNAs, the origin of the amplification occurring from pre-miRNA primers cannot be specifically distinguished. However, this can be identified by comparing the signal from both pre- and pri-miRNA primer sets (41). Pri-miRNA or pre-miRNA expression can then be measured by qRT-PCR as described above for mature miRNAs described in basic protocol 2.

Taqman^™ Probe design for precursor miRNA in qRT-PCR is challenging, because the presence of isoforms is another issue that needs to be carefully considered when designing assays to quantify miRNA. Numerous pre-miRNAs exist as isoforms of nearly identical mature and pre-miRNA, such as Let-7 miRNA isoforms (54). TaqMan^™ MGB probes were used to discriminate 11 of the let-7 family isoforms (55). It is thus often not possible to use SYBR green detection for the PCR primers to discriminate among the various isoforms. However, this issue can be resolved with TaqMan^™ MGB probes that anneal to the precursor to differentiate isoforms (56).

Multiple pri-miRNA isoforms are difficult to discriminate by qRT-PCR, but depending on size differences, can be detected on the same blot by agarose northern blotting (40, 41). Because of the low abundance of pri-miRNAs, at least 10 µg of RNA should be used for this method.

In miRIP experiments, if there is high background in binding, it might be due to several factors. (i) incomplete washing; (ii) non-specific binding to the beads, which is why a preclear step is important for reducing such non-specific binding; (iii) Antibody not being specific enough; (iv) Utilization of too much antibody which may lead to non-specific binding; (v) Too many cells/too much protein in the cell lysate which may also result in increased non-specific binding. To solve these problems, (i) increase the number of washes; (ii) preclear the lysate with beads; (iii) use an affinity purified antibody; (iv) check the recommended amount of antibody suggested according to the protocol; (v) check the recommended amounts of cells and reduce the number of cells/lysates used for the given RIP.

For the best functional analysis of miRNA mimics and inhibitors, since it varies with each cell type, it may be useful to perform titrations to obtain the best concentrations for each cell type.

Anticipated Results

Unlike traditional alcohol precipitation protocol, the use of a mirVana ^™ miRNA isolation kit allows one to effectively recover all species of RNA, including large mRNA and ribosomal RNA with minimum length of 10-mers. This kit can be used for all cell types and tissues, ranging from 10²-10⁷ cultured cells or 0.5–250 mg tissue per kit. It usually yields about 15~20 μg of total RNA. The small RNA purification procedure allows one to efficiently purify small RNA molecules of about ~200 nt and less. It is critical to obtain sufficient amounts of high-quality RNA for experiments including small RNA preparation for sequencing and other miRNA analyses as described above.

SIGNIFICANCE STATEMENT:

This review summarizes the advances in the analysis of miRNA expression, biogenesis, targeting and regulation, which will shed light on the development of miRNA-based immune therapeutics.