library concentrations at various levels (RNA seq)

[Tefina]

Hi,

I am interested in some library preparation details of RNA seq.
Lets assume you deal with GA or HISeq.

I am just interested in the following numbers:

(1) Input Material:
Usually people take 0.1 - 4 ug of total RNA (or, respectively, 100 ng of mRNA). So of what concentration in which volume are we talking about?


(2) Library concentration after amplification (so after purification, fragmentation, first/second strand synthesis, ligation of adapters):
10nM in 10uL .
Would this be a good average estimate?

(3) Library concentration after denaturation and dilution - so when loading it on the flowcell:
7pM in 120uL / lane.
Is this correct?

I would be especially interested in some numbers regarding the TrueSeqProtocol from Illumina.

Thanks a lot in advance,
tefina

[TonyBrooks]

Hi,

I am interested in some library preparation details of RNA seq.
Lets assume you deal with GA or HISeq.

I am just interested in the following numbers:

(1) Input Material:
Usually people take 0.1 - 4 ug of total RNA (or, respectively, 100 ng of mRNA). So of what concentration in which volume are we talking about?

This is in the TruSeq protocol. You can start with 0.1-4µg of total RNA (in 50µL water) or 100ng of mRNA (in <5µL ). We generally start with total RNA towards the top of that range because most of the time RNA isn't limiting, however we produce buckets of library, so we think we could reduce the input if needed.

(2) Library concentration after amplification (so after purification, fragmentation, first/second strand synthesis, ligation of adapters):
10nM in 10uL.

Would this be a good average estimate?

As I said before we generate a large amount of library (concentration is typically > 200nM in about 30µL. This is diluted down to 10nM for storage.

(3) Library concentration after denaturation and dilution - so when loading it on the flowcell:
7pM in 120uL / lane.
Is this correct?

I would be especially interested in some numbers regarding the TrueSeqProtocol from Illumina.

We load 12pM for TruSeq libraries on our GAIIx, but how much you load depends on what works for you and also depends on how you quantify your library.

[Tefina]

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 50µL?

[pmiguel]

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 50µL?

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.

--
Phillip

[TonyBrooks]

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 50µL?

RNA's vary in length so much that it's pointless to estimate a molarity. If you really wanted to, I guess you could do a back of an envelope calculation

According to Ambion M.W. of ssRNA = (# nucleotides x 320.5) + 159.0

Number of moles = Mass (g) / Molecular Weight
Concentration (M) = Number of Moles / Litre

Of which about 2% will be mRNA.

[pmiguel]

Of which about 2% will be mRNA.

But depending on the organism and the tissue it might be 0.01% or 10%.

--
Phillip

[TonyBrooks]

But depending on the organism and the tissue it might be 0.01% or 10%.

--
Phillip

I said it was pointless

[Tefina]

ok, I see your point,
thanks in any case!