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Best method to concentrate RNA/cDNA?


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5 replies to this topic

#1 sran

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Posted 19 February 2009 - 06:03 PM

Hi there,

I am looking for some ideas from the smart people on here... :wacko:

I need to perform a cDNA synthesis and labelling for use on a microarray.
The kit needs 5 ug of RNA in 12 ul to begin with. From my samples, I am lucky to get in total 2 ug of RNA in 20 ul.

So... I am wondering if the best method is to combine several RNA samples then do a vacuum dry and reprecipitate?..But will this increase my chances of RNA degradation? I'm not sure of a good protocol to follow.

Or should I maybe do 3 separate cDNA synthesis reactions using 1.5 ug of RNA in 12 ul , then pool the cDNA at the end, precipitate and resuspend in much less H2O?

Has anyone done this before?

thanks

#2 pcrman

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Posted 19 February 2009 - 07:56 PM

Are your samples precious? If not, I would redo the extraction to get larger amount of RNA in higher concentration, otherwise, do a alchool (ethanol or isopropanol) precipitation. Because this is just the first step of a big experiment, you want to make sure everything is right and optimal. Do check your RNA quality before use it for your microarray.

#3 sran

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Posted 20 February 2009 - 05:42 PM

Are your samples precious?


Hey there,

yes the samples are hard to come by. I have already spent 6 months optimising the RNA extraction method for these type of samples, and this is the most RNA I will get per sample. The quality is quite good.

So, you suggest an ethanol precipitation to concentrate pooled RNA samples - or pooled cDNA samples?

#4 Biog

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Posted 22 February 2009 - 02:35 AM

Are your samples precious?


Hey there,

yes the samples are hard to come by. I have already spent 6 months optimising the RNA extraction method for these type of samples, and this is the most RNA I will get per sample. The quality is quite good.

So, you suggest an ethanol precipitation to concentrate pooled RNA samples - or pooled cDNA samples?


The "best" solution in this case, if your budget permits, is the in vitro transcription kit to amplify your targets. Check for example the Ambion website (or others) to take a look on kits devoted for this purpose.
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#5 molgen

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Posted 22 February 2009 - 03:11 AM

You sould never ever vacuum dry and reprecipitate your RNA. It is very hard to reprecipitate.
There are a lot of ways to concentrate your samples. You can persipitate, bind to a colonna, Dynal Magnetic Beads

#6 Wolverena

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Posted 22 February 2009 - 10:30 AM

If your RNA is very precious, some type of amplification (as suggested earlier) might be worth a try. I don't have much experience with precipitating RNA, so I can't comment much on that subject. A colleague of mine once had a similar problem and used a kit that produces cRNA (RNA made into cDNA and than to cRNA), which amplified his RNA quite a bit. That kit was particular meant for microarray applications.
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