Real-time PCR optimization - (May/25/2007 )
- How does i adjust at Mg and Primer concentration? and While i adjust at Mg and Primer concentration, Which chemical do i adjust at first? or i must do at same time both of them????
- i will study with whole blood. Until i use it, how does i store it?
what dna polymerase do you use? if you are working with a prefabricated qPCR MM you usually don't have to adjust the Mg.
I have attached some literature where you can find in section 2-5 a protocol of primer and probe optimisation.
For storage: EDTA blood should be directly snap-frozen in liquid nitrogen and stored at -80°C. RNase is a very active enzyme...
I will used LightCycler® FastStart DNA Master SYBR Green I kits contain FastStart Taq DNA Polymerase. is Primer set contain Mg contantration?
The Master Mixes normally contain Mg2+. Primers shouldn't. Still it might be necessary to optimize Mg concentration.
I've looked up your PCR master mix and they provide the Mg seperately. starting amount in the MM is 1mM. So first, i would adjust the Mg concentration ranging from 1mM to 5mM using the recommended primer concentration for this (500nM final concentration, I think). Then adjust the primer concentration as mentioned above.
i want to ask another question. i will work cytokine mRNA expesion. in reverse trsncription step, 1st Strand cDNA Synthesis Kit for RT-PCR (AMV)† give two primer, i am confused, which primer is suitable?
thanks a lot for advice.
and while i adjust primer or Mg concentration, what do i use as template? housekeeping gen or sample on my investigation
Random primers amplify all the RNA presented (including rRNA and tRNA if you have them in your RNA sample) and can generate different sizes which is less preferable for real-time I heard. OligoT primers only amplifies from the ends of mRNA, so it's usualy a better choise if you want to amplify a mRNA and it's not wery long (not more that 4 kbs if I remeber it right).
We use oligoT primers for our mRNA expression studies.
Third option is to use a gene-specific primers for RT, if your target is low abundant.
Only thing I noticed on your RT kit - it doesn't specify whether it is suitable for real-time, cDNAs for realtime shouldn't contain RNA-DNA hybrids, usual reverse transcribed templates for classic RT-PCR have. So enzyme used usualy contains Rnase H, and I'm not sure, but AMV is a Rnase H- type.
Roche has a real-time suited RT kit - Transcriptor, but I don't know what the difference is. (price maybe, mostly )
You have two different PCR reactions, one for housekeeping and one for cytokine, yes? So then you must adjust Mg concentration for each of them. Use a cDNA that contains both transcripts and you have a plenty of, from control cells, control blood, etc.
And for a blood storage, we never store blood, but only either purified nucleic acids (if we need DNA) or Trizol lyzate (i we need RNA or both). I don't know how much stable the blood in freezer is, but Trizol in -80 is pretty stable for years, more that RNA itself.