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Manipulating RNA Concentrations/Amounts


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#1 w7u

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Posted 19 March 2011 - 08:44 PM

Hello guys,

Sorry for the very silly questions; my first month working in a molecular lab.

I have few RNA samples and would like to extract the mRNA for cDNA library construction. I can't use the smart cDNA library route as the sequencing center prefers mRNA as an input.

The samples concentrations are 100 ng, 150 ng, 164 ng, 400 ng, 431 ng, 439ng and each one of them is eluted in 30 l TE. I need to pool them in the end to cover the transcriptome diversity from different populations.

The commercial kits I reviewed [Poly(A)Purist, MicroPoly(A)Purist, RiboMinus and Poly A Tract SystemIII) require the following starting materials: 0.2-2mg, 2-400g, 2-10lg and 0.1-1mg, respectively.

I am not sure though how to convert the concentration into these starting materials required by the kits? Does it make any sense to multiple the concentration(mass X volume) by the solution volume and the resulting number would be the mass of my RNA? Can I multiple the total concentrations of all the samples by the total volume of all the samples and think that the resulting number would be the total RNA I have?

Cheers.

#2 pito

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Posted 20 March 2011 - 04:22 AM

Do those kits only state the mass of needed DNA? Dont they say anything about volumes? I mean: how big are the tubes for example?


You have for example a sample with 400 ng in 30l (concentration is thus: 13,34ng/l).
So this is for ex. in the range of the 2-400g startconcentration requirement....

But you say you need to pool them, you mean add them all together?
If this is the case then you will have an end volume of: 30l X 6(samples)= 180 with 100 ng+ 150 ng+ 164 ng+ 400 ng+ 431 ng+ 439ng of RNA.

Wich means in total: 1434ng RNA in 180l or a concentration of: 7,967 ng RNA per l


And then you can start calculating how much l you need to take each time to have a certain amount of RNA (in ng) for the commercial kits.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 phage434

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Posted 20 March 2011 - 05:54 AM

Pito is making the assumption that what you meant in your post when you said:
"The samples concentrations are 100 ng, 150 ng, 164 ng, 400 ng, 431 ng, 439ng and each one of them is eluted in 30 l TE."
that you really meant that you had a total amount of sample of that mass dissolved in 30 ul.

Your statement, at face value, is meaningless. The units of concentration are mass per volume, not mass. Failing to understand and work with this is almost certainly the root cause of your confusion. If you meant that there was 100 ng in 30 ul, then you should have said "concentration of 3.3 ng/ul in a total volume of 30 ul. Equally likely, and the source of the confusion, is that you could equally have meant a concentration of 100 ng/ul in a total volume of 30 ul, with a total mass of 3 ug. Notice that these two are very different, and give dramatically different results.

So, until you figure out and tell us which of these you really meant (and keep it straight yourself) we can't really help you. By then, however, it will likely be obvious to you, as well. Units matter.

#4 w7u

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Posted 20 March 2011 - 06:37 AM

Thanks guys for the answers.

I should clarify one thing: 100 ng, 150 ng, 164 ng, 400 ng, 431 ng, 439ng are the readings I got off the Nanodrop (expressed as ng/ul). By multiplying each number by 30 (the total volume of solution) I get the total RNA in the 30ul TE buffer: (e.g., 100 ng/ul X 30 ul = 3000 ng/ul) does make any sense? From here, how do compare these numbers with those specified in the commercial kits?

Edited by w7u, 20 March 2011 - 08:00 AM.


#5 pito

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Posted 20 March 2011 - 07:56 AM

Thanks guys for the answers. I should clarify one thing: 100 ng, 150 ng, 164 ng, 400 ng, 431 ng, 439ng are the readings I got off the Nanodrop, I think (correct me) these are ng/uL, right?


Phage is off course correct, I assumed it was in 30l ..


When working with the nanodrop, you should see what "values" it states... The "square" where you read the value, should have a measurement next to it: something like ng/l or mg/ml or...
But normally its indeed ng/l..

And like phage allready stated: its important to know if you are talking about mass , concentration or ...

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 pito

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Posted 20 March 2011 - 08:07 AM

Thanks guys for the answers.

I should clarify one thing: 100 ng, 150 ng, 164 ng, 400 ng, 431 ng, 439ng are the readings I got off the Nanodrop (expressed as ng/ul). By multiplying each number by 30 (the total volume of solution) I get the total RNA in the 30ul TE buffer: (e.g., 100 ng/ul X 30 ul = 3000 ng/ul) does make any sense? From here, how do compare these numbers with those specified in the commercial kits?


thats right, Xng/l is indeed 30*X ng for 30l.

! do notice you wrote: e.g., 100 ng/ul X 30 ul = 3000 ng/ul and this is NOT correct: look at it!


About the commercial kits: if it simply states that you need (for example) 200 ng of RNA,then you need to calculate how much l you need to have that amount of RNA..

However: doenst it state anything about volumes? Because for example if you have 1ng per l, you need 200l of volume to have 200ng , if you for example have 400 ng/l, you only need 0,5l...
YOu see what I mean...

I dont know those kits, but its weird they dont mention some sort of volume (range).

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 w7u

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Posted 20 March 2011 - 03:06 PM

My bad; it is 3000 ng of RNA in that particular vial. Thanks a lot matey :)




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