I have done a transcriptome analysis of a medicinal plant and have got few genes for an important metabolite. Now my question is how to choose the right candidate genes for the qRT-PCR?
Do you need to estimate transcription level? or establish copy number?
What is your biological question? It may be easier to help.
I agree with Rohan that the biological question behind the transcriptome profiling should drive the selection of genes for validation by qPCR. The following questions come to my mind in your case:
Regarding the microarray/RNAseq experiment:
1) how good is the quality of the transcriptome analysis (reproducibility between replicates, technical and biological noise, number of genes identified as expressed, correlation to other data sets if available);
2) for the pathway of interest, check if all the essential members show up in your gene expression experiment;
for the design of the qPCR experiment:
3) identify housekeeping genes to use as reference for the qPCR (at least three genes with different levels of expression based on your data);
4) How many genes in total do you want to test by qPCR (consider available resources, as qPCR experiments may scale up and become quite pricy)?
5) Do you expect high biological variability (i.e. you need to include more biological replicates)?
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