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cDNA prepared with varying amounts of RNA


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#1 mandibula

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Posted 30 August 2010 - 03:43 PM

I purified RNA from 16 different samples and got varying amounts, from 500ng to 1ug.
I made cDNA from all RNA I got from each sample (they are precious!). But now I'm not sure how much cDNA to use for the qPCR reactions. should I assume that I got roughly the same amount of cDNA or can I assume that I get twice as much cDNA if I start with 1ug of RNA than if I start with 500ng?

thank you!

#2 Kaioshin

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Posted 30 August 2010 - 06:15 PM

I purified RNA from 16 different samples and got varying amounts, from 500ng to 1ug.
I made cDNA from all RNA I got from each sample (they are precious!). But now I'm not sure how much cDNA to use for the qPCR reactions. should I assume that I got roughly the same amount of cDNA or can I assume that I get twice as much cDNA if I start with 1ug of RNA than if I start with 500ng?

thank you!



Using total cDNA level is one way to normalize your data. However, I believe one of the better ways to do this is to use an internal control - a gene you know doesn't vary in expression between samples, no matter their phenotype/age/whatever. You'd find this type of thing in the literature for your organism. For example, in Arabidopsis, it's been shown that ubiquitin is stably expressed throughout most conditions. You do qPCR for each sample amplifying both your gene of interest and (for this example) ubiquitin. You can then normalize to ubiquitin. If you're experimental results are:
A = 3 and B = 6
Aub = 2 and Bub = 3
you could normalize as such A = 3/2 and B = 6/3, giving:
A = 1.5
B = 2
So, you have a 33% increase in expression of your gene of interest between samples A and B


Anyone feel free to correct me if I've made an error

-Kaioshin

Edited by Kaioshin, 30 August 2010 - 06:16 PM.


#3 mandibula

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Posted 31 August 2010 - 10:57 AM

Thank you for your answer, but I think it wasn't really what I asked.

My qPCR's are optimized and I do relative quantitation against a housekeeping gene.

Until now, I always made my cDNA from 500ng of RNA, and my real time PCR worked fine if I put 4uL of each cDNA reaction into each qPCR reaction.
But this time I made cDNA from variable amounts of RNA, so I'm not sure if I should use less volume of the cDNA made from more of 500ng of RNA or if in the real time PCR small differences won't change the Ct that much and it won't matter anyway because I can normalize with the housekeeping gene.

thanks for your help!

#4 Kaioshin

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Posted 31 August 2010 - 06:41 PM

Thank you for your answer, but I think it wasn't really what I asked.

My qPCR's are optimized and I do relative quantitation against a housekeeping gene.

Until now, I always made my cDNA from 500ng of RNA, and my real time PCR worked fine if I put 4uL of each cDNA reaction into each qPCR reaction.
But this time I made cDNA from variable amounts of RNA, so I'm not sure if I should use less volume of the cDNA made from more of 500ng of RNA or if in the real time PCR small differences won't change the Ct that much and it won't matter anyway because I can normalize with the housekeeping gene.

thanks for your help!



You certainly could vary the amount of template according to the amount of starting RNA, but the housekeeping gene is what's really going to make it work correctly in the end, no matter if you vary the template amount or not. With that being said, it certainly doesn't hurt to try to equalize your template amounts, it could actually help to some degree. I would just use the starting RNA amount, so if you were to use 2ul template from a sample that had 500ng RNA, I would use 1ul for a sample that originally had 1000ng. Of course, there isn't too much need to be perfectly exact in these calculations, ballpark numbers should be more than enough.




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