Identification and Analysis of Small Nucleolar RNAs by Real-Time Quantitative PCR

Abstract

One of the greatest scientific achievements of the twenty-first century is the completion of The Human Genome Project (HGP). Thereafter, we came to know that the human genome codes nearly 2% for making proteins and thus named as coding genes, suggesting the rest of the genome as noncoding or junk. However, research in the past two decades has shown and established that noncoding RNAs are major contributors of regulating and modulating the various function of cells as well as tissues. Noncoding RNAs can be classified as basis of their sizes in two categories, long noncoding RNAs (>200 nt) and small noncoding RNAs (<200 nt). Small nucleolar RNAs (snoRNAs) are part of the small noncoding RNA family and primarily reside inside the nucleus of eukaryotes. Sno RNAs can be divided into two major categories based on their distinguished structure and function; these are C/D box and HACA box snoRNAs. They participate in the posttranscriptional modifications on ribosomal RNAs (r-RNAs), transfer RNAs (t-RNAs), messenger RNAs (m-RNAs), and small nuclear RNAs (snRNAs). Sno RNAs act as guide RNAs to modify other noncoding RNAs by pseudouridylation or 2′O ribomethylation. We discussed in this protocol about one of the widely used techniques for detection and analysis of snoRNAs, i.e., real-time quantitative PCR (RT-qPCR).

1. Introduction

The Human Genome Project, which began in 1990 and concluded in 2003, pioneered the analysis and sequencing of the 92% of human genome [1–3]. Recently, with the advancement of technology, we got to know the full sequence of human genome [4]. Afterward, it was discovered that nearly 2% of the genome encodes for proteins, and the remaining 98% was considered as noncoding. In the past two decades, researchers reported these noncoding RNAs play a major role in regulation of function of cells in various diseases, including cardiovascular diseases (CVDs) [5–7].

In this chapter, we will focus on a specific type of noncoding RNA. Small nucleolar RNAs (snoRNAs) are a class of noncoding RNAs found in the nucleolus of cells that direct chemical modifications of other RNAs, such as ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs) [8]. SnoRNA can be classified into two classes: C/D box (SNORD) or H/ACA box (SNORA), which are characterized by conserved sequences of nucleic acids, or boxes [5]. snoRNAs are known to guide 2′-O-methylation, which is the addition of a methyl group to the 2′ hydroxyl of the ribose sugar in the RNA backbone, and pseudouridylation, the conversion of uridine to pseudouridine. Eukaryotic snoRNAs are commonly found within the introns of both coding and noncoding genes. Additionally, some snoRNAs are transcribed by RNA polymerase II from specific snoRNA loci in the genome [5]. In recent years, snoRNA has exhibited abilities to serve as potential disease biomarkers [5]. Real-time quantitative PCR (RT-qPCR) is a widely used technique for identification and analysis of snoRNAs.

2. Materials

We summarize the procedure necessary to identify snoRNAs in cells and tissues (such as the heart) as follows (Fig. 1).

Fig. 1.

Schematic representations of steps involved in identification of snoRNAs by real-time quantitative PCR

2.1. RNA Isolation and Quantification

• miRNeasy tissue/cells advanced mini kit (Cat # 217604, Qiagen, USA) (see Note 1).

• Ethanol for molecular biology.

• 4 °C microcentrifuge (see Note 2).

• Mini vortexer (see Note 2).

• Accu-jet pro pipette (see Note 2).

• NanoDrop One spectrophotometer.

• Nuclear-free tips including 10 μL, 20–200 μL, 1000 μL (see Note 2).

• Ultrapure nuclease-free water (see Note 2).

2.2. cDNA Preparation and Real-Time PCR

• High-capacity c-DNA reverse transcription kit (Cat #4368814, Applied Biosystems, USA) (see Note 3).

• Mini Amp Thermal Cycler (see Note 2).

• QuantStudio real-time PCR system (QuantStudio 3.0, Applied Biosystems, USA) (see Notes 2 and 4).

• MicroAmp Fast 96-well reaction plate (0.1 mL or 0.2 mL) (see Note 4).

• MicroAmp optical adhesive film.

• Filter pipette tips.

• Microcentrifuge tube.

• PlateFuge^™ Microplate Microcentrifuge with rotor and plate carrier.

3. Methods

In the following protocol, all necessary steps should be performed at room temperature unless stated otherwise.

3.1. Primer Designing

To design the primer specific to any snoRNA, we will follow the convergent method of designing primers.

3.2. RNA Isolation

RNA isolation for identification of snoRNAs can be performed as per requirement of the experiments in the cell cultures or tissues. We outlined the procedures as follows:

1. Follow the manufacturer’s protocol for RNA isolation (miR-Neasy tissue/cells advanced mini kit, Cat # 217604, Qiagen, USA). Before starting the RNA isolation, reconstitute all the buffers as directed in the handbook of this kit. Keep all the buffers at room temperature. This method of RNA isolation will provide total RNA as well as small size RNAs (<200 nt).

2. Homogenize 30 mg of frozen tissue or 1 × 10⁷ cells in 260 μL of RLT buffer. For homogenization of cells/tissues, any process can be used such as syringe needle or tissue rupture II (see Note 5).

3. After homogenization of cells/tissues, add 80 μL of buffer AL (provided with kit) and mix well by pipetting or vortexing. Keep the samples at room temperature for 3 min.

4. After 3 min of incubation with AL buffer, transfer the lysates into a genomic DNA eliminator column (provided with kit) and centrifuge at 8000× g for 30 s. Make sure all the liquids come into the collection tube, if not then centrifuge again.

5. If working with animal tissues, add 20 μL of RPP buffer, vortex it, and keep for 3 min at room temperature (see Note 6). Then, centrifuge for 3 min at 12,000× g to precipitate the pellet. Transfer the supernatant into a fresh 2.0 mL tube.

6. Add 1 volume of isopropanol to the 2.0 mL tube and mix by pipetting. Transfer 700 μL of volume from previous steps to a fresh RNeasy mini column and centrifuge for 30 s at 8000× g. Discard the flow-through and reuse the column and collection tube if using more than 700 μL of volume.

7. In the following steps, add the buffers (RWT and RPE) as 700 μL and 500 μL as per directed by the manufacturer and centrifuge for 30 s at 8000× g. Discard the flow-through and transfer the column to a new collection tube (see Note 7).

8. Add 80% ethanol (500 μL) to the RNeasy mini column and centrifuge at 8000× g for 3 min. Remove the column and keep in another collection tube, and place in centrifuge for 1 min at 15,000× g to make sure there is no more ethanol left in the column.

9. Take out the dry column from centrifuge and put it in a 1.5 mL tube (supplied with kit). Add 25–30 μL of RNase-free water (supplied with kit) in the column and centrifuge for 1 min at full speed.

10. This eluted RNA can be quantified immediately on nano drop or can be stored in a deep freezer (−20 °C or - 80 °C) for long-term storage.

3.3. cDNA Synthesis

1. For cDNA synthesis, carry out the following steps as per directed by manufacturer’s protocol (high-capacity c-DNA Reverse transcription kit (Cat #4368814, Applied Biosystems, USA). This recommended kit can be used up to 2 μg of RNA to make cDNA in 20 μL of reaction (see Note 8).

2. Keep all the components of the cDNA synthesis kit on ice to thaw, except enzyme (see Note 9).

3. Set the reaction volume as per required (we refer a 20 μL of reaction volume) (Table 1).

4. Keep the cDNA synthesis reactions in a thermal cycler (Table 2).

Table 1.

The ingredients and essential amounts required for cDNA synthesis in 20 μL reaction mixture

Ingredients	Amount required (for 20 μL reaction mixture)
10× RT buffer	2.0 μL
100 mM dNTP	0.8 μL
10× random primer	2.0 μL
MultiScribe reverse transcriptase enzyme	1.0 μL
RNase inhibitor	1.0 μL
Nuclease-free water	3.2 μL
Total = 20 μL	10.0 μL + 10 μL of RNA

Table 2.

The incubation of RNA at different temperatures and time for cDNA synthesis

Incubation	Step 1	Step 2	Step 3	Step 4
Temperature (°C)	25	37	85	4
Time (minutes)	10	120	5	Hold

3.4. Identification of snoRNAs by RT-qPCR

1. Synthesized cDNA from the previous section will serve as a template for identification of desired snoRNA.

2. Use snoRNA primers to set up the RT-qPCR (see Note 10).

3. Set up the reaction as listed in Table 3 as per requirement like triplicate or tetraplicate.

4. Seal the plate with an optical adhesive film and spin it in a microplate microcentrifuge.

5. Set the reaction in QuantStudio 3 and load the plate. Select the protocol for SYBR chemistry (see Note 11).

Table 3.

Ingredients of SYBR green chemistry for setting up RT-qPCR

Ingredients	Volume (10 μL/well)	Volume (20 μL/well)
Power up SYBR master mix (2×)	5.0 μL	10.0 μL
Forward primer and reverse primer	0.5–1.0 μM	0.5–1.0 μM
Template cDNA	1 ng–10 ng	1 ng–10 ng
Nuclease-free water	Enough to fill to 10.0 μL	Enough to fill to 20.0 μL
Total	10.0 μL	μL

3.5. Analysis of q-PCR Data

1. Analysis can be performed by utilizing the cycle threshold (Ct) from QuantStudio 3.0, using 2^{e (Δ-Ct)} method for quantification.

2. U6 snRNA primer is widely used as a housekeeping control noncoding RNA (see Note 12).