real-time pcrs not reproducible

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alice.lunardon
alice.lunardon's picture
real-time pcrs not reproducible

Hello, 

I have a very difficult problem to solve with real-time pcrs: I am using dilutions of cDNA pools to test for primer efficiency. The same day I make new dilutions (1:10, 1:50, 1:100,..) I obtain a good standard curve; when I freeze the dilutions and I repeat the same reactions the day after I obtain higher cycles for each dilutions, and they increase if I freeze again my dilutions and repeat the reaction for a third time. It seems that more time passes more my cDNA degrades. The least diluted cDNA (1:10) is the one that is more stable over repetitions. 
I use DNase-free RNase-free water and filter tips. The negative control is clean.
How can I know if I have contamination in my original RNA or cDNA? 
Please if you have any suggestions they would be of great help for me!

Thank you very much!

Alice

Ivan Delgado
Ivan Delgado's picture
Hi Alice,

Hi Alice,

cDNA is not very stable, and if you freeze and thaw it multiple times it will degrade. My recommendation is to aliquot your cDNA into smaller volumes so that you do not have to freeze and thaw it more than once or twice. Ideally you could prepare a bunch of single use aliquots so you would never have to thaw your cDNA more than once.

Hope this helps.

alice.lunardon
alice.lunardon's picture
 Hi Ivan,

 Hi Ivan,

thank you for your suggestions. My colleague uses the same kits that I use to extract RNA and perform retrotranscription and he uses the diluted cDNA multiple times and he sees no difference in the resulting Ct, even after freeze and thawing the cDNA for 10 times. It is like that I know that what I am doing is the standard method in my lab, that must works, so the problem seems not be the choices of the kits or how to store the cDNA, but some problem of contaminations or inhibotors, so related of how I work in specific...but I can't understand where this problem could be!

Thank you very much!!

Alice

Biju
Biju's picture
 Hi Alice

 Hi Alice
It s a well known fact that repeated fereezing and thawing with damage DNA/RNA.I suggest  the following steps.
1. Aliquote a small amount of the diluted cDNA for each day use in several vials.Freeze all.Take one out each day, thaw and use.
2. Alternatively, I have used stored cDNAs, sufficient for 1-2 reactions, at 40C for qPCR with good results.As a last point always ensure thorough mxing of the cDNA before aliqutoing each time for the reaction.
The reason for the least diluted DNA being more stable is just because of the fact of the highest concentration.
Hope this helps.
Biju Joseph

Ivan Delgado
Ivan Delgado's picture
Hi Alice,

Hi Alice,

If your colleague is able to freeze and thaw his cDNA 10 times without seeing degradation then he is very lucky. More than likely your colleague is studying a cDNA that is much more stable than yours.

My recommendation to you is not to think there is a problem with your cDNA but that it is degrading as expected when exposed to multiple freeze and thaw cycles. 

As I pointed out in my previous post the "problem" is freezing and thawing. If you aliquot your cDNA as I suggested, and as Biju suggested too, I believe you will solve your problem.

alice.lunardon
alice.lunardon's picture
 Hello,

 Hello,

I would like to thanks all for the help, I solved my problems in this way as you suggested:

-to test primer efficiency I make serial cDNA dilutions, I use them one day to make 3-4 real-time plates and than I don't use these dilutions any more, because I use also 1:100 and 1:1000 dilutions.
-to perform real-time pcr on my samples I choose the dilution 1:50, make an amount sufficient for two days of experiments and I store it at 4°C during the night.

In this way my experiments were reproducible!

Unfortunately in this way I thaw and freeze the cDNA 1x multiple times, so I think it could degraded sooner, so that I have to re-perform the retrotranscription to have new cDNA 1x.

Alice