Protocol for detection of in vitro R-loop formation using dot blots

Summary

Dot-blot analysis is a technique that allows for fast and convenient detection and identification of nucleic acids and proteins. Here, we provide a guide for nucleic acid isolation from eukaryotic cells and sample processing to detect RNA/DNA hybrids. We then provide detailed steps to quantify dot signal intensity. This protocol can be adapted for screening conditions that result in the accumulation of R-loops.

For complete details on the use and execution of this protocol, please refer to Smith et al.¹

Graphical abstract

Highlights

• •Extraction of genomic DNA from eukaryotic cells
• •RNase III degradation of double-stranded RNA
• •Setup of microfiltration unit and nitrocellulose membrane
• •Chemiluminescent detection of RNA/DNA hybrids and digital quantification

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Dot blot analysis is a technique that allows for fast and convenient detection and identification of nucleic acids and proteins. Here, we provide a guide for nucleic acid isolation from eukaryotic cells and sample processing to detect RNA/DNA hybrids. We then provide detailed steps to quantify dot signal intensity. This protocol can be adapted for screening conditions that result in the accumulation of R-loops.

Before you begin

R-loops are triple-stranded nucleic acid structures that consist of an RNA/DNA hybrid and a displaced DNA strand. R-loops typically arise during transcription as nascent RNA threads back and binds the DNA template at GC rich sequences.² RNA/DNA hybrids are naturally occurring in 5%–10% of the human genome and it has been estimated that 59% of human genes contain R-loop forming sequences.³ Physiologically, R-loops regulate the rates of transcription termination, promote telomere stability, and class switch recombination.⁴ However, increases in R-loop accumulation or dysregulation of their resolution represent an important source of genomic stress and instability. Mutations in R-loop regulatory factors lead to the aberrant accumulation of R-loops and disrupted transcriptional rates have been implicated in a variety of diseases including cancer, ALS, and nucleotide expansion disorders, among others.⁵ Growing evidence also demonstrates R-loop accumulation can underlie the unscheduled induction of inflammatory responses.^1,6 Thus, there is increased interest in understanding molecular processes that promote R-loop accumulation and factors that contribute to their detection and processing.

Given the widespread function across the genome and association with a variety of diseases, the study of R-loops has amplified rapidly since their discovery over 40 years ago.⁷ The discovery and characterization of Ribonuclease H (RNase H), an endoribonuclease specific to RNA/DNA hybrids,⁸ as well as the development of the S9.6 monoclonal antibody,⁹ has improved the study of R-loop structures and formation. Many protocols have been developed to probe R-loop formation in different contexts, both in vivo and in vitro. Here we describe bulk, population analysis of R-loops using the Dot-blot technique.

The Dot-blot is a highly reproducible, high-throughput screening technique to assess global R-loop changes across different cell types and experimental conditions. Dot-blot, and its counterpart Slot-blot, have been successfully implemented in the characterization of R-loop levels across different cell types and has allowed for rapid screening of novel R-loop regulators. Dot-blots rely on isolation of genomic DNA and eventual dotting and cross-linking the nucleic acid onto a nitrocellulose membrane. This facilitates antibody detection of nucleic acid structures that can be coupled with simple chemiluminescent imaging like Western blots. Beyond the use of the specialized microfiltration units, most other materials include general lab equipment and reagents, making the Dot-blot relatively inexpensive and easier to perform compared to other R-loop analysis techniques. This protocol describes the isolation of genomic nucleic acid using silica columns. The detection of RNA/DNA hybrids relies on the use of the S9.6 monoclonal antibody for detection, which selectively binds RNA/DNA hybrids over other double stranded structures. Given the potential cross reactivity with double-stranded RNA (dsRNA), it is important to use RNase III to cleave dsRNA and RNase H to cleave RNA/DNA hybrids. This serves to assess the specificity of S9.6 signals. This protocol is designed to offer rapid, semi-quantitative analysis of R-loop abundance under different conditions which can be completed within two days.

Optimize cell culture conditions

Optimize the seeding density of cultured cells prior to isolating DNA from experimental samples. We use 6 × 10⁵ AC16 cells plated with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 12.5% FBS, 2 mM Glutamine, 100 U/mL Penicillin and 100 mg/mL Streptomycin in 10 cm plates. These cells are maintained at 37°C in 5% CO₂ for 16–24 h prior to harvesting for downstream processing. Optimal seeding numbers and densities should be determined for each cell line/cell type. We find that a seeding density that yields 70%–80% confluency prior to nucleic acid isolation improves consistency across experiments. High seeding densities can disrupt cellular transcription rates leading to variance in R-loop detection across replicate experiments.

The optimal genomic DNA yield is 5–10 μg for each biological replicate with concentrations ranging from 250–1000 ng/μL. The ratio of absorbance at 260 nm and 280 nm (260/280) is used to assess purity of nucleic acids. The expected 260/280 ratio should range between 1.8 and 2.1. Additionally, the 260/230 ratio indicates the presence of organic compounds. Acceptable 260/230 ratios range from 2.0–2.2.

Prepare buffers, isolation kits, and dot blot apparatus

Timing: 0.5–3 h

• 1.
  Start by preparing enough buffers for the required number of samples that will be processed.

  • a.
    Buffers should be used within 3 months of preparation.
  • b.
    Excessive freeze-thaw cycles should be avoided.
• 2.
  One day prior to the start of the experiment, thoroughly clean the microfiltration unit to prevent sample contamination and ensure proper nucleic acid binding.

  • a.
    Wash components of the apparatus with deionized (DI) water.
  • b.
    Spray each component with RNase inactivating (e.g., RNaseZap) solution.
  • c.
    Rinse with 70% ethanol.
  • d.
    Rinse the unit with DI water and allow it to dry overnight.
• 3.If using nucleic acid isolation kits, reconstitute and store reagents per manufacturer’s guidelines.

Note: It is recommended to use nuclease-free water and fresh molecular grade 200-proof (100%) ethanol when processing samples and reconstituting reagents.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti DNA-RNA hybrid (S9.6) (1:2,000 dilution)	Kerafast	RRID: AB_2687463
Recombinant anti-ds DNA antibody (rDSD/4565) (1:2,000 dilution)	Abcam	RRID: AB_3065119
Donkey anti-mouse IgG (HRP-conjugated) (1:10,000 dilution)	Jackson ImmunoResearch	RRID: AB_2340770
Chemicals, peptides, and recombinant proteins
Water, nuclease-free	Thermo Fisher Scientific	A57775
Magnesium chloride (1 M)	Thermo Fisher Scientific	AM9530G
Sodium chloride (5 M), RNase-free	Thermo Fisher Scientific	AM9760G
RNase III	Thermo Fisher Scientific	AM2290
RNase H	New England Biolabs	M0297
RNaseZap RNase decontamination solution	Invitrogen	AM9780
UltraPure 1 M Tris-HCl buffer, pH 7.5	Invitrogen	15567027
Tris, 1.0 M buffer solution, pH 6.8	Thermo Fisher Scientific	J63831.K2
Tris-HCl	Fisher Scientific	T3253-500G
Tris base	Fisher Scientific	BP152-1
Tween 20	Sigma-Aldrich	P2287-500ML
Bovine serum albumin (BSA)	Sigma-Aldrich	A9418
2-Mercaptoethanol	Sigma-Aldrich	M6250
SuperSignal West Pico PLUS chemiluminescent substrate	Thermo Fisher Scientific	34580
Phosphate-buffered saline (PBS)	Gibco	14190-136
Trypsin (0.5%)	Corning	25-052-Cl
Gibco DMEM, high glucose	Thermo Fisher Scientific	11965092
Fetal bovine serum (FBS)	Sigma-Aldrich	F1051-500ML
Penicillin, Streptomycin, glutamine solution	Corning	30-009-Cl
Critical commercial assays
NucleoSpin tissue DNA kit	Macherey-Nagel	740952.50
Experimental models: Cell lines
AC16 cell line	Millipore	RRID: CVCL_4U18
Software and algorithms
Fiji10	Schindelin et al., 2012	https://imagej.net/software/fiji/
Image Lab Suite	Bio-Rad	12012931
Other
0.2 mL, thin wall PCR tubes	Neptune	3735.X
NanoDrop 2000 spectrophotometer	Thermo Fisher Scientific	3042816
ProFlex thermocycler	Thermo Fisher Scientific	4484073
96-well Bio-Dot microfiltration unit	Bio-Rad	1706545
Nitrocellulose membrane, 0.45 μm	Bio-Rad	1620115
Spectroline UV crosslinker	Fisher Scientific	11-992-89
Blot absorbent filter paper	Bio-Rad	1703967
ChemiDoc imaging system	Bio-Rad	1708370
Heat block	Eppendorf	EP5384000020
Rocking platform	VWR	10127-872

Materials and equipment

10× Ribonuclease Buffer (compatible with both RNase III and RNase H)

Reagent	Final concentration	Amount
NaCl [5 M]	1500 mM	0.3 mL
Tris pH 7.5 [1 M]	500 mM	0.5 mL
MgCl2 [1 M]	100 mM	0.1 mL
Water, nuclease-free	N/A	0.1 mL
Total	N/A	1 mL

20× SSC stock solution- pH 7.0 (Wetting Buffer)

Reagent	Final concentration	Amount
NaCl	3 M	175.3 g
Sodium Citrate	300 mM	77.4 g
Water, nuclease-free	N/A	1000 mL
Total	N/A	1 L

Add sodium chloride and sodium citrate to 800 mL of nuclease-free water. Once fully dissolved, adjust the pH to 7.0 using hydrochloric acid (12 N). Bring up the volume to 1 L with nuclease-free water. Dilute to stock solution to 6× SSC with nuclease-free water to rehydrate the nitrocellulose membrane.

10× TBS

Reagent	Final concentration	Amount
Tris-HCl	152.2 mM	24.0 g
Tris base	46.2 mM	5.6 g
NaCl	1500 mM	88.0 g
Total	N/A	1 L

Add Tris-HCl, Tris base, and NaCl to 800 mL of distilled water. Once fully dissolved, adjust the pH to 7.6 using hydrochloric acid (12 N) or NaOH. Bring up the volume to 1 L with distilled water. Dilute to 1× when making TBS-T solution.

Membrane Stripping Buffer

Reagent	Final concentration	Amount
Sodium dodecyl sulfate (SDS)	2%	2.0 g
2-Mercaptoethanol	100 mM	700 μL
Tris pH 6.8 [1 M]	50 mM	5 mL
Total	N/A	100 mL

Add SDS, 2-mercaptoethanol, and Tris pH 6.8–80 mL of distilled water in a chemical fume hood. Once dissolved, bring the volume up to 100 mL with distilled water. Before use, heat up to 50°C in a warm water bath in a chemical fume hood. Cover membrane with stripping buffer in a blot box.

Other Solutions

Name	Reagents
TBS-T	1× TBS + 0.1% Tween-20 (v/v)
5% w/v BSA in TBS-T (membrane blocking solution)	Dissolve 5 g of BSA in 100 mL of TBS-T. Filter with a 0.22 μM filter to ensure all BSA is dissolved in solution.

Alternatives: Other spin column based genomic DNA isolation kits, such as the DNeasy Blood & Tissue Kits (QIAGEN), can also be used for nucleic acid isolation. It is important to ensure that sample processing does not include treatment with RNA nucleases as these could alter the stability of RNA/DNA hybrids. Although column-based DNA isolation yield consistent results across samples, you may also use organic solvents (phenol:chloroform:isoamyl alcohol) for DNA extraction. A Proteinase K treatment step is still recommended to remove the viscosity of the sample and improve isolation of DNA.

Nylon membranes can be used in place of nitrocellulose membranes for sample immobilization. Nylon membranes have a higher affinity for nucleic acids as well as increased binding affinity for shorter lengths of DNA as compared to nitrocellulose. Because of this increased affinity, it is more likely to obtain a higher background signal during chemiluminescent imaging (step 47). We did not directly test differences between nitrocellulose and nylon membranes in the use of this protocol.

Step-by-step method details

Day 1 (part 1): Cell culture and sample collection for genomic DNA harvest

Timing: 2–3 h

Genomic nucleic acids can be derived from cells in culture or tissues. We describe the culture conditions of AC16 cardiac muscle cells¹ and DNA extraction using commercially available kits.

• 1.
  Culture cells in complete growth media (described above).

  • a.
    At the time of harvest, transfer spent media to a 15 mL conical.
  • b.
    Wash cells twice with 1× PBS to remove traces of media.
• 2.
  Lift cells with 2 mL of trypsin and incubate for 5 min at 37°C.

  • a.
    Use the saved spent media to quench trypsin.
• 3.
  Pellet cells by centrifugation at 800 × g for 5 min and discard media.

  • a.
    Wash cell pellets twice with ice-cold 1× PBS.
• 4.To extract nucleic acids, we followed the Cultured cells protocol listed in the NucleoSpin Tissue DNA kit from Macherey-Nagel. Refer to the manufacturer’s user manual for a detailed extraction protocol.¹¹

Note: If you use a different DNA purification kit that calls for RNA nuclease treatment step, forego this step. This will lead to degradation of RNA/DNA hybrids.

• 5.Elute nucleic acid from the Spin-Columns in 50 μL of pre-warmed elution buffer.
• 6.Repeat step 5 with elute material to improve DNA yields.

CRITICAL: Before the nucleic acid extraction, pre-warm the elution buffer to 56°C in a water bath. Avoid the use of elution buffers containing EDTA which interferes with downstream ribonuclease digestion. Alternatively, use pre-warmed nuclease-free water.

• 7.Quantify the genomic DNA concentration for each sample using a UV-vis spectrophotometer or a similar technique.

Pause point: The genomic DNA can be stored at −20°C for several months, but it is recommended that the DNA concentration is verified after freeze-thawing.

Day 1 (part 2): Ribonuclease treatment of isolated nucleic acid samples

Timing: 20–24 h

Many commercially available genomic DNA extraction kits do not include RNA digestion steps leading to appreciable RNA contamination. The S9.6 antibody cross reacts with highly abundant double-stranded (ds) RNA species.^12,13 To enrich for the specific detection of RNA/DNA hybrids, a dsRNA-specific nuclease, RNase III, digestion step is necessary. A fraction of the samples is digested with RNase H to hydrolyze RNA bound to DNA and serve as a control for the specific detection of RNA/DNA hybrids. An example Dot-blot Ribonuclease Calculation Table (Table S1) is provided to easily equalize DNA concentration and reaction volumes to ensure uniform digestion across samples.

• 8.
  Dilute 5 μg of isolated DNA in 1× Ribonuclease Buffer in a clean, RNase-free PCR tube (0.2 mL).

  • a.
    10× Ribonuclease Buffer should be thawed on ice and reagents kept cold throughout the protocol.
  • b.
    10× Ribonuclease Buffer should be kept as 100 μL aliquots and store at −20°C for up to 3 months.
  • c.
    PCR tube strips provide easier handling and better temperature control during incubation and deactivation steps of ribonuclease digestion.
• 9.Add 1 Unit (U) of RNase III per μg of genomic DNA (total 5 U).
• 10.Incubate samples at 37°C for 2 h in a thermocycler.
• 11.Heat inactivate RNase III at 65°C for 20 min.
• 12.
  Aliquot half of the reaction into a new, clean, RNase-free PCR tube strips (0.2 mL).

  • a.
    Adjust final volume to 50 μL, adding 1× Ribonuclease Buffer (5 μL). See critical note below.
• 13.
  Add 10U of RNase H/μg of DNA (total 25 U) to the remaining sample.

  • a.
    Adjust final volume to 50 μL and add 1× Ribonuclease Buffer (5 μL). See critical note below.

CRITICAL: We find that total reaction volumes of 50 μL yielded the most consistent processing of RNA. It is important to replenish the ribonuclease buffer prior to RNA/DNA hybrid digestions with RNase H. Failure to do so decreases the enzymatic activity of RNase H.

• 14.Incubate both sample sets from steps 12–13 at 37°C for 16–20 h.
• 15.Heat inactivate RNase H at 65°C for 20 min.

Day 2 (part 1): Immobilization of nucleic acid samples onto nitrocellulose membrane

Timing: 4–5 h

Immobilization of nucleic acids on nitrocellulose membranes allows for detection of specific nucleic acids using antibodies followed by chemiluminescence.

• 16.Remove samples from thermocycler and place on ice.
• 17.
  Prepare serial dilutions of undigested and RNase H digested samples in a 96-well plate.

  • a.
    Adjust the final volume to 100 μL using nuclease free water.
• 18.Incubate samples on ice while the microfiltration unit is assembled.

Note: We recommend starting dilutions of 500, 250, and 125 ng/100 μL, but optimal dilution can be determined empirically for distinct cells and tissue types. We recommend probing the same membrane with nucleic acid antibodies. Alternatively, you load technical replicates to assess reproducibility and/or to incubate the membrane with distinct nucleic acid antibodies if necessary.

Assembling the dot-blot microfiltration apparatus

• 19.Align the gasket support plate into position in the vacuum manifold.
• 20.Place the sealing gasket on top of the support plate and roll the sealing gasket several times into the gasket support plate (Figure 1A).

Note: Align the openings in the sealing gasket with those in the support plate. This supports optimal vacuum pressure and prevents the formation of halos on the Dot-blot.

• 21.
  Pre-cut the nitrocellulose membrane to fit within the sealing gasket and sample blotting device.

  • a.
    The recommended membrane size for a 96-well device is 8.5 cm × 12 cm. If smaller membrane must be used, then seal off unused wells using wax films.

Note: Handle the nitrocellulose membranes carefully using gloves and clean, dry tweezers and scissors to avoid bending or contaminating the membrane. Using a pencil, mark the orientation of the membrane. Alternatively, cut a notch on the corner of the membrane to track sample loading and orientation.

• 22.
  Prewet the nitrocellulose membrane using 6× SSC membrane wetting solution.

  • a.
    Gently rock the membrane in a clean blot box for 10 min.
  • b.
    Store SSC at ambient temperature (23°C–24°C) and discard solution if it becomes cloudy.
• 23.Remove the membrane from the wetting solution and drain excess liquid by dabbing the membrane on a filter paper.
• 24.
  Place the membrane on the sealing gasket in the apparatus, covering all sample wells (Figure 1A).

  • a.
    Remove air bubbles using a blotting roller.
• 25.Place the sample template on top of the membrane and partially tighten the securing screws.

Note: To achieve even pressure across the apparatus, partially tighten the screws following the indicated order (Figure 1B). Do not tighten the screws entirely.

• 26.Attach vacuum flask tubing to the flow value in the vacuum manifold.
• 27.Turn on the vacuum and set the 3-way valve to setting 1 (Figure 1C).
• 28.With vacuum on, firmly tighten the screws following the indicated pattern (Figure 1B) to create a seal between the sample template and the membrane.
• 29.Adjust the flow valve so that the vacuum manifold is open to air pressure- setting 2 (Figure 1C).
• 30.
  Transfer 100 μL of nuclease-free water to each of the 96 wells using a multi-channel pipette to rehydrate the membrane.

  • a.
    Use sterile tips and buffer reservoirs to simplify this process and avoid contamination of the membrane.
  • b.
    During this step, the flow valve must be kept in setting 2.
• 31.Apply vacuum to the apparatus by adjusting the flow valve to setting 1 (Figure 1C).
• 32.Once the nuclease-free water drains from all the wells, adjust the flow valve to setting 2 to expose the unit to air (Figure 1C).

Note: It is important to rehydrate the membrane and to ensure that the vacuum is turned off after re-hydration. This prevents excessive drying of the membrane which may cause uneven spotting or halo formations. Monitor wells closely to ensure even flow of nuclease-free water through the membrane across all wells. If sample filtration across any well is slow or uneven, avoid using these well of the sample plate.

CRITICAL: The recommended loading volume for the microfiltration unit used in this protocol 100 μL. Lower volumes may cause uneven loading and the appearance of halos. If the number of samples being loaded onto microfiltration unit is less than the 96 wells, fill the remaining wells with 100 μL of nuclease-free water. Alternatively, close off unused wells with wax film. Other microfiltration devices might differ in loading capacity.

Figure 1.

Microfiltration unit setup and operation steps

(A) Assembly order for microfiltration unit.

(B) Stepwise sealing screw tightening.

(C) Flow valve setting to control vacuum into the microfiltration unit.

Sample immobilization and UV cross-linking

• 33.
  Load samples from step 17 onto the microfiltration unit using a multichannel pipette.

  • a.
    If technical replicates were performed in step 17, load technical replicates in separate wells of the Dot-blot sample template.

Note: Avoid generating bubbles and break up any air pockets that may have formed during sample loading.

• 34.
  Open the flow valve setting to setting 1 (Figure 1C) and wait until all wells have drained.

  • a.
    If loaded sample wells are not draining, gently pipette the sample up and down a few times to relieve any bubbles that might have formed.
• 35.
  Once samples have filtered, loosen the sealing screws in the opposite order used for tightening.

  • a.
    Keep the vacuum on and maintain the flow valve in setting 1.
• 36.Quickly, lift the sample template from the membrane and gasket below and set aside.
• 37.Lift the nitrocellulose membrane from the sealing gasket.
• 38.Place the membrane on top of a dry, clean filter paper.
• 39.Immediately place the filter paper with the membrane in the UV crosslinker chamber and cross-link nucleic acids unto the membrane at an energy of 1,200 mJ x 100/cm².

Day 2 and 3 (part 2): Detecting nucleic acids by immunoblotting

Timing: 16–20 h

This step describes the incubation of nitrocellulose membranes with antibodies that recognize RNA/DNA hybrids (S9.6) and double stranded DNA followed by incubation with secondary antibodies conjugated to horseradish peroxidase (HRP). Chemiluminescent detection of HRP signal is performed after incubation.

Note: Perform blocking, antibody incubations, and wash steps on a rocker.

• 40.Remove the membrane from the UV crosslinker and trim the membrane using sharp, clean scissors if necessary.

Note: If technical replicates were loaded onto the membrane, they can be used to measure technical accuracy, or the membrane can be cut for incubation with distinct antibodies.

• 41.
  Block non-specific antibody binding by pre-incubating the nitrocellulose membrane with 5% BSA in TBST (blocking buffer) for 30 min.

  • a.
    Store TBS at 23°C–24°C.
• 42.Remove the blocking buffer and wash the membranes twice with TBST for 5 min.
• 43.Incubate membranes with primary antibodies diluted in 5% BSA in TBST overnight (16–18 h) at 4°C. Rock gently.

Note: The recommended starting dilution of purified S9.6 antibody is 1:2000. The recommended starting concentration of purified dsDNA antibody is 1:2000. The optimal antibody concentrations can be further optimized for each cell type and experimental treatment.

• 44.Remove the primary antibody and wash the membrane twice for 5 min using TBST.
• 45.Incubate membranes with HRP-conjugated secondary antibody diluted 1:10,000 in 5% BSA in TBST for 1 h at 23°C–24°C.
• 46.Remove the secondary antibody and wash the membrane twice for 5 min each with TBST.
• 47.Develop the membrane using enhanced chemiluminescent reagent according to manufacturer’s instructions.
• 48.Digitally capture luminescent images using a Bio-Rad ChemiDoc Imaging System or similar detection system.

Note: Take several sequential images of both membranes incubated with S9.6 and dsDNA antibodies. This will provide a range of images to optimize signal quantification. High sensitivity chemiluminescent substrates may be used for detection of low levels of R-loops in samples or samples with low signals. Membranes can be used to probe with S9.6 antibody followed by probing dsDNA. It is important to strip the primary/secondary antibodies prior to staining with an additional antibody.

• 49.
  Optional: Incubate the membrane in 10 mL Stripping Buffer at 50°C for 30 min. Rock the blot box every 5–10 min.

  • a.
    Store Stripping Buffer at room temperature (23°C–24°C) for up to 3 months.
• 50.Discard the stripping buffer and wash the blot three times for 5 min using TBST. Rock the blot during washes.
• 51.Repeat steps 41–48 to probe and re-image the membrane.

Day 3: Digital image analysis

Timing: 1–2 h

This step describes image analysis to calculate signal intensity of RNA/DNA hybrids (S9.6) and loading controls (dsDNA). This permits the derivation of a signal ratio to assess changes in R-loop frequencies across samples.

• 52.Load raw digital image files to FIJI.

Note: TIFF files are the preferred file format as most of the pixel information in conserved for analysis. Avoid the use of JPEG files as lossy compression can change pixel intensity values.

• 53.Change the bit-depth of the image to 8-bit. Go to image → type → select 8-bit.
• 54.Select the optimal blot image in which the most highly concentrated samples for both S9.6 and dsDNA blots have a gray intensity pixel range from 100–200 (See note).

Note: To determine the gray intensity value for a particular dot, go to analyze → set measurements→ check mean gray value. Use the oval selection tool to highlight each dot. Go to analyze→ measure.

CRITICAL: Avoid images with saturated pixel, distortions, streaks, or halos.

• 55.
  Once images are selected, determine the rolling ball radius in pixels.

  • a.
    Make sure that the unit of length is set to pixel. Go to analyze → set scale → unit of length: pixel.
  • b.
    If the unit is not in pixels, type “pixel” into the unit of length textbox and enter.
• 56.Using the line tool to measure the diameter of each nucleic acid dot in the image and record the rolling ball radius value in pixels (Figure 2A).

Note: The rolling ball radius is commonly the length of the largest objects of measurement in an image. Determining the rolling ball radius is critical for performing rolling ball background subtraction. This will remove spatial variations in the background of the two images, standardizing the background for analysis.

• 57.
  To subtract the background, go to process → subtract background → rolling ball radius.

  • a.
    Input the value determined with the line tool in step 56.
  • b.
    Make sure light background is checked.
• 58.Invert both images by selecting image → lookup tables → Invert LUT (Figure 2B).
• 59.
  Set the measurements criteria in ImageJ for quantifying dots.

  • a.
    Go to analyze → set measurements → check Integrated Density leaving all other boxes unchecked.
• 60.
  Using the circular region of interest tool, circle the first dot to be quantified.

  • a.
    Make sure the area encompasses the entire dot.
  • b.
    This becomes your region of interest, or ROI (Figure 2C).
• 61.
  Use the shortcut “ctrl + m” (“command+ m” on Mac) to measure the ROI.

  • a.
    Record the integrated density value.
  • b.
    Use the same ROI for each subsequent dot measurements.

CRITICAL: Do not generate new ROIs. Click and drag the circle from the previous dot to the next, centering the ROI on the dot being measured.

• 62.Normalize the S9.6 signal intensity (measured by the integrated density value in step 59) over the corresponding dsDNA signal intensity for each sample. These values can be normalized across replicate experiments to changes in R-loop formation across treatments (Figure 2D).

Figure 2.

Workflow of Dot-blot digital analysis for signal quantification

(A) Digital image selection, (B) background subtraction and image inversion, (C) ROI selection for density measurement, and (D) plotting of signal ratios.

Expected outcomes

RNA/DNA hybrids are readily detectable in undigested samples following S9.6 antibody staining (Figure 3A left, upper panel / RNase H-). This signal decreases in a dose dependent manner across serial dilutions. S9.6 reactivity is greatly diminished in samples that have undergone enzymatic digestion of this substrate by RNase H treatment (Figure 3A right, upper panel / RNase H+). Incubation with dsDNA antibody is used to demonstrate equal loading across samples left undigested and those treated with RNase H (Figure 3A lower panel). Again, signal intensity decreases across serial dilutions. Differential S9.6 staining should be seen across samples of equivalent nucleic concentration in samples where R-loops are stabilized or processed. Below is sample data derived from AC16 cells in which the DEAD box helicase 41 (DDX41) has been deleted via CRISPR-Cas9 genome-editing technologies. DDX41 is known to resolve transcriptional R-loops¹⁴ and thus, its depletion leads to increased RNA/DNA hybrid detection via Dot-blot (left, upper panel-compare DDX41 KO with H1). H1 are non-targeted control cells that serve to establish baseline levels of R-loops. Treatment with RNase H resolves RNA/DNA hybrids and diminishes S9.6 staining. Quantification of signal ratio indicates a 2-fold increase in cells that lack DDX41 (Figure 3B).

Figure 3.

Example of Dot-blot and signal quantification

(A) Example of RNA/DNA hybrids detected in wild-type (H1) and DDX41-deficient AC16 cells.

(B) Signal density calculation of the dot-blots at 500 ng DNA concentration. Figure 3A is reprinted with permission from JR Smith et al., 2023.¹

Limitations

The Dot-blot assay allows for rapid, controlled screening of the pathophysiological role of R-loops and the cellular factors involved in their formation or resolution. However, this assay does not define the subcellular localization of RNA/DNA hybrids. Transcriptional R-loops form in the nucleus⁴ or the mitochondria.¹⁵ Importantly, disruption in endonuclease expression has been shown to result in the extrusion of genomic RNA/DNA hybrids to the cytosol.⁶ Immunofluorescent approaches can probe RNA/DNA hybrid formation in situ.¹ Antibody based RNA/DNA hybrid pull-downs constitute other R-loop profiling methods that can address regions of R-loop formation. These techniques provide greater targeted or genome-wide resolution when coupled with PCR (qDRIP) or massive parallel sequencing (DRIP-seq¹⁶). The S9.6 antibody described in this protocol can be used for both the immunofluorescence and immunoprecipitation methods.¹⁷ Finally, the Dot-blot provides qualitative changes in R-loop levels that can be semi-quantified through densitometry. Because it relies on chemiluminescent signal produced by the secondary antibody for quantification, the true concentration is not a direct measurement of R-loop formation itself and cannot be a quantifiable method of R-loop levels in the cell. Other methods including DRIP-seq and qDRIP have tried to resolve this issue using spike-in controls^18,19 allowing for normalization and absolute quantification between samples.

Troubleshooting

Problem 1

Low DNA yield or poor DNA quality (as determined by UV spectrometry).

Potential solution 1

Low DNA yields can be caused from a variety of conditions. Commercially available genomic DNA extraction kits have a maximum starting material that can be isolated, usually around 10⁶–10⁷ cells. If during any of the spin isolation steps, liquid is not fully flowing through the column, the filters may be clogged preventing binding and elution steps. The proper number of cells should be determined empirically if DNA isolation is an issue (step 1).

Improper preparation of the isolation kit’s components may also cause insufficient DNA yields (step 4–6). Make sure that proteinase K is used and prepared properly as the lysis step contains many contaminating proteins that can clog the filter. Increasing the Proteinase K digestion time may improve yields.

Low DNA yields and residual phenol or guanidine contamination lead to low 260/280 or 260/230 ratios. If using column-based purification, complete washes and column drying as indicated to avoid reagent carry-over into the eluted nucleic acid. Ensure that the elution buffer is free of contamination.

Problem 2

Insufficient ribonuclease digestion (determined by S9.6 antibody signal in step 48).

Potential solution 2

The ribonuclease enzymes, RNase III and RNase H, most commonly fail because of expiration dates or too many freeze thaws cycles. These enzymes are stored at −20°C in a freezer that does not have self-defrost cycles to prevent temperature fluctuations. Utilize a microcentrifuge tube freezer block to prevent temperature fluctuations while working at the bench.

Both RNase III and RNase H require a divalent metal ion for its enzymatic activity.^20,21 We found the buffers supplied from the commercially available enzymes to be incompatible for sequential digestion. Thus, we use a universal 10× Ribonuclease Buffer described above which supplies the necessary cofactor metal ions for catalysis. Replenishing the buffer yielded consistent RNase H activity (step 13).

Problem 3

Unequal DNA signal across samples (determined by dsDNA antibody signal).

Potential solution 3

Unequal loading is likely a result of nucleic acid quantification (step 7), sample evaporation or pipetting errors during sample processing or loading onto microfiltration unit. When using PCR tubes or tube strips, check that the lids are closed entirely to prevent evaporation during sample incubation (steps 10–15). Use calibrated pipettes to carefully transfer samples.

Changes in signal intensity can also derive from uneven filtration of the sample in the device. Creating a tight seal on the microfiltration unit with the tightening screws helps maintain even flow across wells (steps 25–28). The formation of air bubbles can also disrupt the flow of sample (step 33). These can be dislodged by gently pipetting up and down samples from slow draining wells using a p200 micropipette.

Problem 4

Halo formation around nucleic acid dots.

Potential solution 4

Halos may form around the edges of dotted nucleic acid. These halos may confound qualitative and quantitative assessment of dot intensities. Make sure to rehydrate the membrane after unit setup (step 29–32). Keep the flow valve at setting 2 as the samples are being loaded (step 33). This prevents the drying of the nitrocellulose membrane which can affect nucleic acid binding.

Additionally, over and under tightening of the sample template to the vacuum manifold can cause halos formation. Adjust the tightness of your microfiltration unit to prevent bending the membrane or leakage from sample template (step 28).

Problem 5

Weak or no signal.

Potential solution 5

The absence of chemiluminescent signal may arise from a variety of conditions:

Degradation of RNA/DNA hybrids. The nuclease treatment steps are followed incubation at 65°C for enzyme inactivation (steps 11 and 15) are frequently used during RNA/DNA hybrid detection^1,14 as previous studies examining the effect of temperature in RNA/DNA hybrid immunoprecipitation demonstrates that RNA/DNA hybrid stability is not affected.²² On the other hand, the studies determined that sample incubation with broad RNase A, a non-specific nuclease commonly used during DNA extraction, can diminish RNA/DNA detection. Avoid RNase treatment during genomic DNA isolation (Step 4).

Dry membranes result in poor nucleic acid binding. The nitrocellulose membrane must be soaked in 6× SSC buffer prior to setting up the microfiltration unit and prior to sample loading (step 22).

Sample leakage can lead to diffusion of dot signal. This can be prevented by creating a tight seal for each sample well (steps 25–28).

Failure to properly cross link nucleic acid to the nitrocellulose can results in low signals. The recommended energy for crosslinking is 1,200 mJ x 100/cm² (step 39). As different UV crosslinkers may have different conversion rates in their Energy Mode interface ensure that the energy settings are input properly. Ensure that UV bulbs are functioning properly and calibrated to deliver accurate quantities of energy.

The S9.6 and dsDNA antibodies should be kept at −20°C before use. Additionally, re-used antibody dilution could account for weak or no signal. Freshly diluted antibody solutions and empirically optimized antibody concentrations can overcome weak signals (step 43).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Adriana Forero (Adriana.forero@osumc.edu).

Technical contact

Further technical inquiries should be directed to the technical contact, Adriana Forero (Adriana.forero@osumc.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new unique code.