Hey ladies and gentlemen! I’m using qPCR to investigate how gene expression changes under different conditions in bacteria. I did RNA extractions of the bacteria under the desired conditions and converted it to cDNA using the iScript cDNA synthesis protocol. Our thermocycler malfunctioned towards the end of the reaction, so I need to verify that I actually have the cDNA I want and not just a failed reaction.
My question is: can I set up a conventional PCR using the cDNA as a template, and use primers for a bacterial housekeeping gene, run the PCR product on a gel and hope to verify that cDNA is present in that way? I would prefer not to do it with qPCR if possible, because sybr green is expensive! My concerns are (1) that using the same 2-step protocol with the same temperatures and number of cycles won’t work because the supermix I’m using for regular PCR is slightly different than the one for qPCR, and (2) the amount of amplified DNA will be too small to visualize on a gel because ethidium bromide is way less sensitive than sybr green. Before doing the cDNA synthesis, I normalized the RNA to 0.44 ng/µL as per the instructions, which is very small compared to the amount of template that a person would normally use for PCR. Can I remedy this by doing a lot of cycles (like 50+) for my PCR?
Or is this idea doomed to fail, in which case should I just bite the bullet and run qPCR on my unverified cDNA? Thank you in advance!