How to deal with and combine the inconsistent results?
I tried to take control of my sample loading & reaction set up. As a result, I got relatively consistent expression of my internal control among plates (I used HPRT, Ct ~20).
I used to normalize the results by using sample1, and combined the normalized data from each plate together. In this way, I get accepted degree of deviations.
But I realized that this way is problematic, and I should use a SINGLE sample for all the rest of my data. The problem is: when I tried to combine all of my sample1 from each plate, I noticed there were huge inconsistency.
eg. in terms of gene A expression, the delta Ct of mouse 1 &2 may be 2&4.
This is my exaggeration, the inconsistency is not that big, but still outstanding. Biological replicates are necessary, but the inconsistency drives me crazy.
Is there anyway to fix it?
Hi Zhitao,
If I understand correctly, you are running multiple PCR plates and need to analyze data across plates, correct? What do you mean by use a single sample for the rest of your data?
I would suggest analyzing each plate individually, expressing the data as fold-change from the average of the control group (on that plate) using the delta delta Ct method. Then combine the fold-change values from each plate for groupwise analysis. This method will help to control inter-run variability. Using the same control samples in each plate will also help.
Dear Zhitao, you can use DeltaDeltaCt method for relative quantification. Another way is the software of your Real-Time PCR system especially if you are using Roche LC, advanced relative quantification option can analyse and normalize your Cp results with respect to housekeeping gene as a calibrator.
Best regrads.
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