Hi Everyone,

I'm quite new to RNA work but have been working with it the last while and been having some issues. I extract RNA and RT to cDNA and aim to sequence to see the effects of specific variants.

I've been running some samples recently and continuously am not seeing any bands on my gel, I've tried different samples and new primers to no avail and was hoping someone could give some advice. When I have extracted RNA from fibroblast cells I have got amazing yields and after RT these produced super clear and strong bands on the gel which I was able to send for sequencing and get clear results (My RNA yield for these were around 2000ng/ul and added between 0.8ul-1.2ul for generating cDNA).

I've been recently working with RNA extracted from muscle and blood which obviously have generated much lower yields of RNA (between 10 and 90ng/ul) but I have not got any good results from these. For the RT I have tried using 1ul, 5ul and 15ul of RNA from these samples on a couple of different attempts and when I measure the cDNA the concentration seems okay (for example in one instance was 900ng/ul) and following this I have ran gels using dilutions of 1:2, 1:5 and 1:10. On a couple of different times I have been able to see VERY faint bands but adjusting the dilutions to try and compensate isn't helping at all.

Not sure what else to try to improve these results but if anyone could provide any insight I would be very grateful

Thank you