I am trying to denature RNA samples made up of stem-loop structures and dumbbell elements, it has been a really difficult task, I already did RT-qPCR in two steps and I could not even then. Does anyone know how I can make cDNA from these samples, please.
It might be worth trying the Rt step in the presence of 1Molar betaine ( final concentration) which is very good for relieving secondary structure in DNA and is also very kind to enzymes
You can also try using the OneScript Hot Reverse transcriptase, or the OneScript Hot cDNA synthesis kit. The OneScript enzyme can work at much higher temperatures, up to 80'C. Hopefully this can solve your secondary structure issue.
Thanks to all, I tried with Betaine 0.5M, 1M, DMSO and UREA, changed the primers, individually and still not successful. I'll look up the OneScript Hot enzyme to give it a try, also plan on doing it again in two steps with a SuperScript IV and let you guys know how it goes.
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