I have inoculated rice agar plates with A. oryzae, extracted RNA and synthesised cDNA from it, and run the samples together with a DNA ladder on a 10.6% polyacrylamide gel. I then stained them using SYBR™ Safe DNA Gel Stain and view them under U.V light. However no bands are visible and only the DNA ladder shows. However when I run the same RNA samples on a 1% agarose gel with ethidium bromide, the RNA bands are visible. What could be the reason for my P.A.G.E not to work?
First of all why do you need to visualize the cDNA? I will rather use other quantitative methods or downstream processes e.g., RT--real-time PCR to verify the cDNA stability. In my experience, running cDNA on gels only show you smears rather clear band.
Atul Pandey
I wanted to visualise cDNA to check if my primers were valid. i later ran a RT PCR on the cDNA and I was able to view bands on the agarose gel. However, I cant explain why I had no RNA bands on my polyacrylamide gel
Are you using a gene-specific primer during for your cDNA synthesis (i.e. not random primer or oligo-d(t)) ? If not, there is no reason to run the cDNA on a gel before doing the RT-PCR.
How much RNA input was used for the cDNA reaction?
A few other things to consider:
- Some SYBR dyes are much more effective at detecting double stranded DNA or single stranded DNA (cDNA is a ssDNA). I couldn't find the details on SYBR Safe, but I don't think this is the primary issue.
- The cDNA will run faster than expected due to being single stranded. Is it possible it ran off the gel?
Yes i was using a gene-specific primer since I was investigating the presence of particular genes.
Unfortunately, I'm not sure of the RNA concentration since I don't have a nanodrop, but 2ul of my extracted RNA after being dissolved in RNA free water.
cDNA running of the gel could be an explanation however I still cant understand why my RNA was visible on an agarose gel and not on a polyacrylamide gel.
I would test your SYBR Safe dye on the agarose gel as well, just to rule out the possibility that is the culprit.
If that is not the problem, I think it's probably a technical error (e.g. Perhaps there was not enough loading dye in your samples and they floated out of the wells).
Troubleshooting these things remotely can be difficult. If there is someone else with experience doing RNA PAGE in your lab, it might be useful to have them observe them next time you perform a PAGE.
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