Dear Colleagues and Mentors,
I'm trying to determine the expression of some Genes with SYBR Green Quantitative RT-PCR protocols. But, I'm facing problems to formulate the standard curve. I tried with many different combination and adjustments like changing primer concentrations, temperature etc. Would be better if anyone suggest me any ideas or recommendations.
Cheers!!!!
I generate my standard curve by using a dilutions of plasmid containing the specific gene product.
Hope this helps.
What specific problem(s) are you seeing? Low efficiency? Poor reproducibility?
In general, I've seen three main problems:
1) primers that are not efficient. You can try adjusting the annealing temp, primer concentrations, etc. BUT that only works well if you are very close to 100% efficiency and need to make a slight adjustment. Honestly, you will probably need to design and test several sets of primers to find ones that work well.
2) math/pipetting errors in creating the dilution for the standard curve. Triple check your math, pipette carefully, make a large volume and aliquot samples for use. Freezing and thawing will degrade your standard DNA.
3) math/pipetting erros in setting up the qPCR reaction. Make a master mix, pipette VERY carefully, briefly centrifuge to remove air bubbles and make sure you don't get fingerprints on the plate seal.
Also, make sure you don't repeatedly freeze and thaw your qPCR mix. If it starts to degrade, you will get inconsistent and low amplification results.
@Katie A Clark: Thanks for you details explanation and suggestions. I'm also thinking the same and will do as you mentioned.
@Naga Rama Kothapalli and Mikael Kubista: thanks for the suggestions.
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