RNA dot blot protocol
Licia Miller Product Manager
RNA dot blotting is a technique similar to Northern blotting; however, RNA samples cannot be separated by electrophoresis and require membranes for imaging .
Materials required
Reagents :
- RNA sample
- RNase-free water
- TBST (1×TBS, 0.1% Tween-20)
- blocking solution
- Primary antibody
- Secondary antibody (goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP)
- ECL substrate
- Streptavidin-Alexa Fluor®488
- Methylene blue staining buffer(0.2% Methylene blue in 0.4 M sodium acetate and 0.4 M acetic acid)
Equipments：
- RNase-free tubes
- Heat block
- Positively charged nylon membrane
- 10 cm plastic petri dish
- SG Linker (with 254 nm bulb)
- Imaging system
Alexa Fluor® is a registered trademark of Life Technologies.
Experimental steps
1. Remove the RNA sample from the refrigerator (aliquot) . Thaw the RNA sample tube and place it back on ice.
2. Using RNase-free tubes, gradually dilute the RNA sample with RNase-free water.
2.1 The final concentration for reference: 2.5/0.25/0.025/0.0025 μg/μL.
2.2 We dilute the positive oligonucleotide containing RNA modification and the negative oligonucleotide without modification to 50 ng/μL and 5 ng/μL.
3. Incubate serially diluted RNA in a heating block at 95°C for 3 minutes to disrupt secondary structure.
4. Immediately after denaturation, place the test tube on ice to cool down to prevent the re-formation of RNA secondary structure.
5. Cut the positively charged nylon membrane to the appropriate size, which needs to be large enough to accommodate the 2×4 grid.
5.1 Use a pencil to lightly mark the grid on the membrane to assist with sample addition .
5.2 Transfer the membrane to a clean 10 cm plastic Petri dish.
6. Use a pipette to mix the RNA sample by pipetting up and down. Drop 2 µL RNA onto the membrane.
Notes: Avoid touching the membrane with the pipette tip. Allow the pipette droplet to diffuse onto the membrane via surface tension. Change the pipette tip after each loading, including between batches of the same sample .
7. Immediately transfer the dish containing the membrane into the chamber of the SG Linker.
7.1 Remove the lid of the culture dish and ensure that the RNA side of the membrane faces upwards.
7.2 Crosslink RNA to the membrane using UV light: irradiate at 254 nm for 30 min-1 h .
8.Wash the membrane with 10 mL TBST (1×TBS, 0.1% Tween-20) for 5 minutes at room temperature, shaking gently to wash away unbound RNA.
9. Place the membrane (RNA side down to prevent the membrane from accidentally drying out) in 10 ml of blocking buffer and incubate with gentle agitation at room temperature for 1 hour.
10. Discard the blocking buffer and incubate the membrane with the primary antibody (at the given dilution, usually 1 μg/mL working IgG) in 10 ml of blocking buffer overnight at 4°C with gentle shaking .
11. Flip the membrane so that the RNA side is facing up and wash the membrane three times in 10 mL TBST with gentle agitation for 10 minutes each time.
12. Invert the membrane so that the RNA side is facing down and incubate the membrane with the appropriate secondary antibody (goat anti-rabbit IgG-HRP or anti-mouse IgG-HRP) in blocking buffer with gentle agitation for 1 hour at room temperature .
13. Invert the membrane so that the RNA side is facing up. Wash the membrane four times in 10 mL TBST with gentle agitation for 10 minutes each time .
14. Incubate the membrane with ECL substrate for 5 minutes .
15. Expose the membrane using an imaging system. Take images in high-resolution mode with exposure times ranging from 1 second to 3 minutes at appropriate intervals. After exposure, wash the membrane with TBST for 10 minutes.
Notes: 1) If the oligonucleotide is biotinylated, probe with Streptavidin-AlexaFluor® as a loading control.
2) Ensure that the previously imaged ECL has been quenched;
3) Clean as thoroughly as possible to reduce background .
16. Incubate the membrane with Streptavidin-AlexaFluor® 488 (in TBST) in a black box for 1 hour .
17. Transfer the membrane to the imaging system. Take images using the Alexa 488 fluorescence mode .
18. After fluorescence imaging, transfer the membrane to 10 mL of methylene blue staining buffer (0.2% methylene blue in 0.4 M sodium acetate and 0.4 M acetic acid). Incubate for 30 minutes with gentle agitation.
Tips: For loading controls of non-biotinylated RNA, methylene blue or ethidium bromide can be used.
19. Rinse the membrane with dH2O until the background is clear (approximately 30 seconds to 60 seconds).
20.Image the methylene blue stained membrane using an imaging system .
For more product details, please visit the Aladdin Scientific website.