how to dilute cDNA from mRNA concentration? - (Apr/15/2008 )
I have cDNA reversed transcribed from mRNA. I didn't take the mRNA concentration until after I finished reverse transcribing the cDNA, so now each cDNA sample has a different concentration. How do I dilute the cDNA samples so they are of equal concentration (if I only know the mRNA concentration)?
sample / RNA concentration ng/ul
sample1 / 32.1
sample2 / 28.5
sample3 / 30.7
11uL of mRNA used with 1uL oligodT, 1uL dNTPs, 4uL 5X First-Strand buffer, 2uL 0.1M DTT, 1uL Superscript II RT
i'm not sure i have understood what you mean. Do you mean you know the RNA concentration before RT? If so,
the first sample's RNA concentration is 32.1 ng/ul and you took 11ul, so you used 353ng for RT. Generally people assume a 100% conversion RNA to cDNA (measuring cDNA is not practical) so your cDNA's concentration is 17.65ng/ul (in 20ul after RT). When you know all your sample's concentration you can diluite cDNA at your convenience.
Hope this helps
reverse trancribe will give different efficiency and rate of reverse transcribe. so the concentration will be different even you use same amount of starting mRNA.
Just to be safe, why not take the reading of your cDNA concentration?
as ros75 suggests, it is usually considered a 100% efficiency. it all depends on what you're using your cDNA for. if it's only checking whether your gene is expressed or not then it should be ok but if you want to compare the differential expression of the same gene amongst different samples i'd do the extractions again.
As toejam said, if you are planning to measure gene expression, I would repeat the RT again (not necessarily the RNA extraction). For quantitative studies is not a good idea to use different amounts of starting RNA, as the RT efficiencies vary in function of that and you could get false results, do your RT using equal amounts of RNA.