I'm currently doing RT-qPCR and I have a problem of poor reproducibility across replicate samples. I looked at my results and it's not one replicates that cause the problem but it varies between the gene I'm doing. So one replicate can have a great reproducibility for one gene and for the one a poor reproductibility . Any advice on how to get better reproducibility across replicate samples.
Have you checked whether your pipette is still o.k. or that the tips are fitting?
Do the problems occur always at the same position of your plate? In this case the thermocycler might have a problem with temperature uniformity.
I've had this problem before and it was to equipment calibration. After recalibrating it worked just fine.
Any reason why you are doing a single techical replicate? This approach may be fine if you are having a quick glance at Ct values and to check for primer dimer formations. Also if you have a microarray/NGS this is approach is permissible.
RT-qPCR can be very sensitive to pipetting inaccuracies.
For data quantitation I have always used the triplicate approach as occasionally a biological replicate would show a variation in Cts. Remember a cycle of variation means a 1-fold change which is a lot.
Although it takes more time and reagent, requiring me to use 2X 96 well plates, using 3 technical replicates resulted in more robust data.
Well I have 4 sample for each condition that I do that in duplicates.
- Badges
- Science topic