On real time PCR, cDNA kits etc.
Hi there
A couple of (good) news for people dealing with real time PCR:
1-The Mj machine at the SBM can read a signal from a 8ul reaction. That means you can set your reaction using only 4ul of SybrGreen/replicate. However you really need a good pipette since small pipetting errors become critical at this point.
2- The QuantiTect cDNA kit from Quiagen is so far the least expensive and more efficient kit I had the chance to try. There is only one caveat: you need RNA already digested with DNAse. The kit includes a "genomic DNA wipeout reagent" to take care of genomic DNA before the cDNA synthesis, but as far as I can tell it's useless. Scaling don the reaction works fine, the kit works like a charm and it is really a pleasure to use (it's a 2 step process that only takes about 30').
1-The Mj machine at the SBM can read a signal from a 8ul reaction. That means you can set your reaction using only 4ul of SybrGreen/replicate. However you really need a good pipette since small pipetting errors become critical at this point.
2- The QuantiTect cDNA kit from Quiagen is so far the least expensive and more efficient kit I had the chance to try. There is only one caveat: you need RNA already digested with DNAse. The kit includes a "genomic DNA wipeout reagent" to take care of genomic DNA before the cDNA synthesis, but as far as I can tell it's useless. Scaling don the reaction works fine, the kit works like a charm and it is really a pleasure to use (it's a 2 step process that only takes about 30').
Hope this is helpful
Till next one
Gabriele Amore
Etichette:
technical stuff
2 commenti:
hey !
A comment on the last post. Thanks !
Enrique.-
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Comment on “On real time PCR, cDNA kits etc.”
Really good to know! BUT I’ll stress the point
of pipeting small volumes and the big potential
variability that comes with it. Be careful and include two (three?) replicates.
The only point I don’t agree with is on how
useless it can be to use the ‘genomic DNA wipeout
reagent’. Are you saying that you don’t use it
because you have checked that there’s no DNA
contamination after RNA extraction?
I’ll say that it depends on how I do the
extraction (i.e. Kit, eurozol, etc..). But it
applies even if I trust the 2 step extraction kit
(DNA and RNA) of the QuantiTect cDNA kit from
Quiagen. Unless I want to run always a control on
my qPCR plate with RNA in stead of cDNA, the
DNAse step should be done. Not to include that
control can be quite problematic in order to be confident on positive results.
An then, what happens if I actually find a
positive result on my RNA control? Re-do the
whole cDNA? In my case I usually have a low yield
of RNA, so I can’t manage to use it directly as a control material.
My two cents on the subject. At the end all
cookers have their special recipe…right?
Cheers !
Enrique Arboleda
______________________________
Enrique Arboleda
Biochemistry and Molecular Biology Laboratory
Stazione Zoologica "Anton Dohrn"
Villa Comunale 1 (80121)
Napoli - ITALY
e-mail: enrique.arboleda@szn.it
Tel. (+39) 081 583-3255
Fax (+39) 081 764-1355
Well as I already said, 8ul is a small volume so it makes your experiments to be more error prone (hence the need for replicates). to stay on the safe side I do 10ul rxs and it's been working very well.
As for the Genomic DNA wipeout, I still use it, just beacuse it's in teh kit and it takes 2 min to do it. However when prepping DNA with quiagen columns (this is how I do it nowadays), I do have to do a DNAse step during the procedure (even if it is considered optional with the all prep DNA/RNA kit). So the DNA wipeout reagent alone is not enough to emove all genomic DNA from my samples. So in my experience it is "insufficient" more than "useless".
Gabriele Amore
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