RNA extraction of a very, very small quantity of tissue

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Tapi
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RNA extraction of a very, very small quantity of tissue

Post by Tapi » Tue Nov 21, 2006 1:19 pm

Hi I have started with RTPCR 2 months ago. I have performed well the experiment but the problem is that now I have to make a RNA extraction from a tissue of "ONLY 0.02 mg" and I have no idea of how I can process this amount of tissue.
If someone could give me some idea I would be very grateful.
Thanks a lot in advance

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LilKim
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Post by LilKim » Thu Nov 23, 2006 6:17 am

hmm... can you pool samples? So that you have a greater total weight?

I used to do trizol extraction off mouse hippocampal tissue... and we often pooled hippocampi of several mouse 'siblings (that underwent the same experimental treatment... the same day... )

buena suerte
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Post by Tapi » Thu Nov 23, 2006 9:11 am

Hi
Unfortunately, this is my bigger problem, I have very limited samples so I only can use this small amount for each experiment

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LilKim
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Post by LilKim » Thu Nov 23, 2006 4:56 pm

Wow.. sorry to hear!

Well, Hmm I'm not sure if there is a reliable-kit that you could get (because i've never used one).

All I can sugggest is find some other tissue and practice extracting RNA from a tissue of a comparable size... and see how it works out.

I don't do RNA extractions very often, so I usually have a low yield my first few times... so, maybe you can just practice to see how much RNA you can extract and wait until you're comfortable... and then do the real experiment.


Wishing you luck!
- KIM

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Post by G-Do » Thu Nov 23, 2006 6:12 pm

Hey there,

Have you already got the RNA? If so, you can amplify it indefinitely using one an mRNA-amplification kit, like the one available from Ambion:

http://www.ambion.com/catalog/CatNum.php?AM1750

The messageAmp kit will turn a small sample of mRNA into a nigh-limitless amount. I have used it with great success in my own research (going from 100ng of total RNA to upwards of thirty micrograms in a single reaction). But the downside is, it's very expensive.

If you haven't got the RNA yet, you might be in a tough spot. I typically run my cells through a QiaShredder RNA-collection kit from Qiagen, then amplify with the messageAmp kit. How many cells do you have in your 20 micrograms? If memory serves, the Qiagen kit needs at least 10^5, or something like that.

Hope that helps.
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Post by Tapi » Mon Nov 27, 2006 9:29 am

Hi:

I think that the problem of using an RNA amplification kit is that I want the RNA for RT-PCR and I must amplify a control gene like beta-actin to standarize results, and I think that this type of kits amplify preferently the most abundant transcripts so the results wouldnt be valid. Have you heared something about this?

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Post by G-Do » Tue Nov 28, 2006 6:40 am

Hi Tapi,

You said:

"...and I think that this type of kits amplify preferently the most abundant transcripts so the results wouldnt be valid."

That is not categorically true. Some amplification kits are better than others, and new protocols are often put on the market without strong QC (meaning, they do have some transcript biases). However:

A while back, the gene expression people at UPenn pooled their resources and did an analysis of one of the popular protocols (Ambion, the one I use most often, and since I'm at UPenn that's probably no surprise). You can judge the results for yourself (the paper is free):

http://physiolgenomics.physiology.org/c ... l/13/2/147

If you want independent verification of these findings, here's what you do. Many big universities have their own microarray facilities, staffed by technicians who have hands-on experience with the experimental nuances of the amplification protocols. Try googling "Harvard microarray facility" or "UPenn microarray facility," get the contact info for those techs, and ask them about it. If - after you've gotten multiple opinions - you feel comfortable taking the risk, go for it.

Hope that helps!
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