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How many samples at a time?

How many samples at a time? - Real-Time PCR and Quantitative PCR Forum

How many samples at a time? - Real-Time PCR and Quantitative PCR Forum


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Old 09-11-2010, 04:30 PM
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Default How many samples at a time?



Hey guys!

I am curious - how many samples do you process for RNA extraction (Trizol) at a time?

Most people that I know don't do more than 10....

I want to do about 16 at a time, keep homogenizing and keep the homogenized ones on ice

What do you think? Good idea, bad idea?
Can it make RNA degrade?

THanks!
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Old 09-11-2010, 05:58 PM
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Smile Re: How many samples at a time?

Quote:
Originally Posted by Anais1979 View Post
Hey guys!

I am curious - how many samples do you process for RNA extraction (Trizol) at a time?

Most people that I know don't do more than 10....

I want to do about 16 at a time, keep homogenizing and keep the homogenized ones on ice

What do you think? Good idea, bad idea?
Can it make RNA degrade?

THanks!
With Trizol, do as many as you want.

I'd handle large RNA extraction with Trizol reagents like this.

Homogenize samples (usually 1ml homogenate generated).
Centrifuge
add 200uL homogenate to 800uL Trizol, invert to mix.
Once those homogenates hit the trizol there is no degradation.
So they can sit around while the rest of the samples are processed. For the very last sample, I time 10 minutes sitting in Trizol reagent, and proceed with the extraction.

I wouldn't put the homogenate in ice, I'd do the extra step of adding/mixing it into Trizol.
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Old 09-11-2010, 07:59 PM
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Default Re: How many samples at a time?

Thanks for the fast response, Danfive!

See, in our lab we homogenize samples in a different way - we place frozen tissue in 1 ml of Trizol and then homogenize. So pretty much if you do about 6 samples, 5 of them will sit on ice for some time in Trizol. So if I will be doing like 16 samples, they would end up sitting on ice for over an hour...this is a concern
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Old 09-12-2010, 05:33 AM
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Default Re: How many samples at a time?

If I'm not mistaken, the components in Trizol will inhibit the activity of RNAses. So therefore leaving your samples (properly homogenized, of course) in Trizol on ice shouldn't be a big concern. However if you are leaving your unprocessed/unhomogenized samples just like that on ice, that will definitely lead to RNA degradation.
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Old 09-12-2010, 09:07 PM
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Default Re: How many samples at a time?

Thanks Joneave,
the samples which go to Trizol are frozen :-)

I haven't decided yet what will I do this week - I may do these samples in 2 days just to be on a safe side

Thanks for the reply!
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