Please define "the right candidate".

Do you need to estimate transcription level? or establish copy number?

What is your biological question? It may be easier to help.

Dear Woody,

In the process of transcriptome sequencing and analysis we would have come across several genes of different functions.

Please suggest me which genes should I select out of theses for the qRT-PCR

Dear Massimo,

I have done a digital gene expression analysis and have got an idea about the expression pattern of few genes. But i wanted to validate these genes (pathway genes) using a RT-PCR so is it good to take the ones analysed or look out for other genes ???

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)?

If it is for checking the pathway definitely pick a gene which is the first step committed for that metabolite only, as many precursor metabolites can be used for other pathways/metabolites