Hi Everyone:
I have a question regarding to qPCR. I am trying to do RT-PCR, and I want to have equal amount of input DNA for my RT-PCR, so I normalize my DNA input based on qPCR values of a reference gene. But when I do RT-PCR using the primers of the reference gene and normalized DNA input, I always got obviously unequal amount of DNA after amplification on agarose gel. My PCR-cycle is 25, and it should be within the linear amplification range.
Does anyone have any tips or tricks that could help me better normalize my input DNA based on qPCR results?
Thank you!!
Why do you normalize your input DNA based on qPCR results?
If you want to have an equal amount of input DNA for your RT-PCR, you should quantify your samples in nanodrop and use the corresponding volume from each one to have the same DNA concentration.
Why do you normalize your input DNA based on qPCR results?
If you want to have an equal amount of input DNA for your RT-PCR, you should quantify your samples in nanodrop and use the corresponding volume from each one to have the same DNA concentration.
Why are you bothering to run out your samples on a gel? The important information from qPCR is the Ct value.
I think you need to talk with someone about how to do qPCR. The "normalization" is using the expression of your reference gene as a way to compare the expression of your gene of interest between samples/treatments.
You do not need to have an equal amount of starting template in each reaction. In fact, if you gene of interest has a really low copy number, you might have to increase the amount of starting cDNA so it's within the detection limit of your assay for those wells.
Hi Yang Zheng, I agree with Daniela and Katie. You should be able to normalise your target gene expression levels to that of the house-keeping gene whether you run them on a gel or in qPCR machine. A usual confunder in qPCR experiments is genomic DNA contamination during the RNA extraction step or that the house-keeping gene is not suitable for your research purpose. Your qPCR oligos will still anneal and amplify the same target genes in gDNA. One way to troubleshoot this is to treat your RNA samples with DNAse before RT-qPCR. Hope this helps.
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