I have run 3 times of RT-PCR for one of my sample. However, fold changes always inconsistant in these three attempts. What is the possible factors contribute to this inconsistancy?
Lim,
Several factors may contribute to PCR inconsistent results. But, if you're using the same sample for these three analysis (1 extraction for the same sample), the same PCR conditions and same reagents, this problem is probably related to pipetting. PCR is a very delicate protocol. When I have this kind of problem I try to increase the final volume of the reaction since small final volumes leads to bigger chance of errors.
Hi Lim,
As real time pcr is highly sensitive method to study gene expression and quantification, even minor deviation from the normal is easily identified. Generally while working with RT-PCR source of variation starts from growth stage / treatments given to test sample. Then second stage at RNA isolation and quantification. Last is pipetting errors. First problem can be eliminated by keeping biological replicates. Second problem can be reduced by keeping technical replicates.
The problems related to quantification can be minimized by quantifying RNA using Qubit kit of invitrogen. If you have used same sample in 3 of your experiment then major reason is pipetting error.
I agree with the above answers.. RT-PCR is very sensitive to small changes including pipetting errors and variability between experiments. One more thing that you may check is the sealing of your samples. high temp in PCR with improper sample sealing may affect final product because of sample evaporation. Also good mixing of your ingredients is crucial for perfect reaction. good luck
re u using one step RTPCR or two step? i mean is your source template cDNA or RNA? to avoid errors try using cDNA saples for most of your experiments as it more stable than the RNA. keep an aliquot of desired concentration ready with u n use the same for all experiments. finally the quality of your cNDA or RNA also matters. in case of RNA integrity is also an issue. repeated thawing may degrade the RN. try to keep working stock at -20 degress instead of repeatedly thawing main RNA sample. since RT PCR is a very sensitive technique lot of precautions have to be take. hoope this helps.
good luck
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