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Old 11-08-2011, 09:29 AM   #4
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Quote:
Originally Posted by Tefina View Post
can you also tell me something about the concentration of the total RNA in the beginning? So assuming that each mRNA has an average length, can you tell me more or less the concentration (nM) in this 50ÁL?
Rule of thumb: 1 ug of 1kb DNA is ~ 1 trilllion (10^12) molecules.

Twice that for single stranded molecules.

But, remember that total RNA is largely rRNA. I would guess the Illumina TruSeq RNA prep libraries yield about 1% polyA+ RNA. Maybe 10 ng per 1 ug of input. So, you are down to about 20 billion molecules.

Then you fragment those molecules. This causes an increase in the number of fragments, but also a lot of those fragments are lost during purification.

Call it a wash. Still at around 20 billion molecules. Then conversion to cDNA. You will lose at least half. 10 billion.

So that is your library really. Sure you will almost certainly do enrichment amplification -- this can increase the number of molecules by >1000x.

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Phillip
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