Hello everybody,
I'm struggling a lot with my RNA dot blots to detect m5c level. Briefly the protocol I followed (based on this paper"Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot” PMID: 38180832")
- Extraction of RNA using Quiagen Rnaeasy mini kit, getting good quality RNA from sh-sy5y cells.
- Denaturation at 65 degrees for 5 minutes
- Loading till 2000 ng of RNA on a positive charged membrane (Amersham™ Hybond™-N+)
- UV Crosslinking
- Blocking in 5% drymilk PBS-Tween (0.1%) 1 hour at RT
- Wash 3 times in PBST
- Incubation with Primary Ab (ab214727 Rabbit monoclonal) diluted 1/500 ON at 4 degrees
- Wash 10 times (x 5 minutes) in PBST
- Incubation with Secondary Antirabbit Ab (1/5000)
- Wash 10 times (x 5 minutes) in PBST
- Incubation with ECL substrate for 1 min and explosion
I used also some samples (RNA standard with known level of m5c methylation I had from an ELISA kit) that are the only in which I can detect some signal, although the high background, while the white spots correspond to my samples. I thought I had lost my RNA during the experiment, but at MB staining you can see that it s still there.
Could you help me somehow? Any suggestion?
Thanks a lot!
Marcello
One possibility is that there is too much template bound to the membrane. Then at the addition of the ecl reagents there is too much activity too soon so by the time you measure the chemiluminescence the reaction has all happened so there is no measurable signal leaving just a background which is actually lower than the surrounding membrane. You could try serial dilutions of your template as a check if this is the case
Dear Paul,
thanks for your kind reply. As you suggested I tried with different dilutions of the samples (also on the membrane in the picture), but without any significant difference in the detected signal.
Thank you again for your interest
Best,
Marcello
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