I want to know the reverse-transcription efficiency with enzymes either from avian myeloblastosis virus (AMV) or from Moloney murine leukaemia virus (MMLV) in RT-PCR or first-strand synthesis of cDNA. For example: if the started mRNA is ~10^6 molecules, how many mRNA will be reverse-transcripted into cDNA?
AMV will produce higher yields, MMLV (especially modified one to work at 48-50oC) will produce better quality. If you use specific oligo or oligo dT I would recommend MMLV. If you are using rnd 6-mers either will work.
If you are using the RNA for NGS RNAseq LC the quality (eg RIN) is not that important for the success, but may affect the transcription profile.
In order to calculate the efficiency of RT reactions, you can follow these steps:
1-Obtain a calibration curve with a DNA standard using SYBR Green (quantitative real time PCR). You will get a linear equation of Ct vs. log of number of cDNA copies.
2-Perform the RT reactions with the RT enzymes you want to test and a RNA standard with the same sequence as the DNA standard. Then, do the QPCR as in previous step.
3-RT Yield is = (copies cDNA/copies RNA)*100. With Ct values obtained using RNA standard, you can calculate copies of cDNA and then get the yield for each RT enzyme.
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