N6-Methyladenosine (m6A) (D9D9W) Rabbit mAb #56593

RNA Dot Blot Protocol

A. Buffers and Reagents

1. 20X Saline Sodium Citrate (SSC) Buffer: 3.0 M NaCl, 0.3 M Sodium Citrate, pH to 7.0.
2. 10X SSC Buffer: Dilute 20X SSC buffer 1:2.
3. 4X RNA Denaturing Buffer: 16.4 M Formamide, 2.8 M Formaldehyde, 26.6 mM MOPS Buffer (6.7 mM Sodium Acetate, 1.3 mM EDTA, 1.3 mM EGTA).
4. Nuclease-Free Water: (#12931)
5. Blotting Membrane: This protocol has been optimized for positively charged nylon membranes.
6. 96-Well Dot Blot Apparatus
7. 10X Tris Buffered Saline with Tween^® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH₂O, mix.
8. Nonfat Dry Milk: (#9999)
9. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
10. Bovine Serum Albumin (BSA): (#9998)
11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
12. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076).
13. Detection Reagent LumiGLO^® chemiluminescent reagent and peroxide (#7003) or SignalFire™ ECL Reagent (#6883)

B. Dot Blot

Note: This protocol is written for spotting either purified total RNA or poly A-purified mRNA (titration of 2 μg, 1 μg, 500 ng, 250 ng, 125 ng, 62.5 ng, and 31.25 ng) onto a positively charged nylon membrane using a 96-well dot blotting apparatus. Depending on the source of the RNA, more or less RNA may be required for detection with the antibody.

Before Starting:
• RNA is sensitive to degradation by RNases, which can affect sample integrity. It is recommended that all surfaces and equipment undergo RNase decontamination.
• Purify total RNA and/or mRNA from cell pellet using an RNA isolation kit. Assess total RNA quality by gel electrophoresis on a 1% agarose gel. The 28S and 18S RNA should present as distinct bands. Smearing indicates RNA degradation. See Figure 1.
• Cut a piece of nylon membrane to fit the size of the dot blot manifold.
• Wet nylon membrane with 10X SSC Buffer.
• Dry membrane by placing it in a 96-well dot blot apparatus and applying vacuum.

1. Dilute RNA to 160 ng/μl in 50 μl of nuclease free water. Denature RNA by adding 16.5 μl of 4X RNA Denaturing Buffer and incubate at 65° C for 5 min.
2. Add 66.5 μl of 20X SSC buffer and immediately chill on ice for 5 min.
3. Add 67 μl of nuclease-free water to bring DNA solution to a final volume of 200 μl with a RNA concentration of 40 ng/μl.
4. Set up a series of six, 2-fold dilutions by adding equal volume of the RNA solution, starting with the RNA solution in Step 3, to nuclease-free water. This will generate seven RNA samples with concentrations of 40, 20, 10, 5, 2.5, 1.25, and 0.625 ng/μl.
5. Apply 50 μl of each of the seven dilution samples into separate wells of the 96-well dot blot apparatus, leaving the last well for nuclease-free water only. The amount of RNA added to each well should then be 2 μg, 1 μg, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 0 ng respectively. Apply gentle vacuum pressure to draw solution through the membrane. Nylon membrane should be mostly dry before Step 6.
6. Remove nylon membrane from the 96-well dot blot apparatus and wrap in plastic wrap.
7. UV cross-link nylon membrane at 1200 J/m².
8. Repeat Step 7 for a second round of UV cross-linking.

C. Membrane Blocking and Antibody Incubation

Optional: To normalize sample loading using methylene blue, apply stain before Section C, Step 1 and capture an image. Rinse blots three times for 5 min each with 15 mL dH₂O. Stain does not affect antibody binding or detection.

1. Incubate membrane in 25 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
2. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
3. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the antibody product datasheet) in 10 ml primary antibody dilution buffer, with gentle agitation overnight at 4°C.
4. Wash three times for 5 min each with 15 ml of 1X TBST.
5. Incubate membrane with the species appropriate HRP-conjugated secondary antibody (#7074 Anti-rabbit IgG, HRP-linked Antibody or #7076 Anti-mouse IgG, HRP-linked Antibody) at 1:2000 in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
6. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
7. Proceed with detection (Section D)

D. Detection of RNA

1. Incubate membrane with 10 ml of LumiGLO^® (0.5 ml 20X LumiGLO^® #7003, 0.5 ml 20X Peroxide and 9.0 ml purified water) or 10 ml SignalFire™ #6883 (5 ml Reagent A, 5 ml Reagent B) with gentle agitation for 1 min at room temperature.
2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap, and capture images using a chemiluminescent-sensitive detection method (film exposure or digital imager).

NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr.

Figure 1. Representative image of isolated, intact total RNA. 28S and 18S RNA should migrate as distinct bands. If RNA presents as a smear, the sample may be degraded and unfit to use in downstream assays. Lane 1 is NEB 100 bp DNA ladder and lane 2 is total RNA isolated from 293T cells.