pulling cDNA - a big error? - (May/10/2006 )

Let's say we have two groups: treated (8 mice) and untreated (8 mice). I want to pull cDNA and make 2 samples: control (untreated) and treated, instead of 16 samples (each containing cDNA from individual mouse). Would it be a big error?

I would not do it, unless you are absolutely 100 % sure every sample has same amount of cDNA (which is never in my case). You see, if you pool (?) the cDNA, you dont take into account the amount differences. Simple example:

total cDNA, "target" cDNA, percentage
100 ug, 5 ug, 5%
80 ug, 6 ug, 7.5 %
120 ug, 12 ug, 10 %
If you pool this you get like 7.7 % or so. Hope it makes some sense, it does to me kinda.

MASSIVE ERROR!

Much better to analyse separately then average the results. That way you can be sure of standard deviations etc

wouldn't do it! even though 8 mice have been treated they could have differing reaction to the treatment, analysing the data individually will allow you to recognise any outlying data points that would otherwise skew your readings!

take the extra time and get reliable data

Thank you all for your replies.

I thougt about measuring the concentrations before pulling the cDNA.

But you're right, it will be more reasonable to make every sample individually to compare individual differences.

if it were ok to pool the cDNA, you wouldn't have to use more than one mouse...for the same reason, see?

This question came to my mind while reading this:
is there the same problem with pooling the RNA samples, before doing the RT? Measuring RNA concentration and mixing same amounts. In this way you eliminate the error due to the efficiency of RT reaction. But, is it enaugh?
Well, I guess that to be able to pool without error you would need a bigger number of samples, right?


coco