Primers for qPCR validation in non-model RNA-Seq

[lzsph]

Hey guys,

Once we got differentially expressed (DE) genes using RNA-Seq, we need to do qPCR validation.

1.For a non-model plant, among the DE genes, maybe most of them are unknown. If you pick some known and unknown genes/transcripts to do qPCR validation, how do you design primers for those unknown (maybe novel) genes/transcripts? (qPCR primers span introns)
2.Generally, how many genes should be selected as candidate genes?


Any suggestions would be greatly appreciated.
Thanks.

Sincerely,
lzsph

[HESmith]

1. You could design a number of PCR primers at discrete intervals (0, 150, 300, 450bp) along the transcript, then test in pairwise combinations using genomic DNA vs cDNA as template. Any pair that produces bands of different sizes (gDNA > cDNA) spans an intron (you can confirm by Sanger sequencing the PCR products).

2. It really depends upon the goal of your qPCR experiment. Remember to include a couple of non-differential genes as controls, though.

[lzsph]

1. You could design a number of PCR primers at discrete intervals (0, 150, 300, 450bp) along the transcript, then test in pairwise combinations using genomic DNA vs cDNA as template. Any pair that produces bands of different sizes (gDNA > cDNA) spans an intron (you can confirm by Sanger sequencing the PCR products).

2. It really depends upon the goal of your qPCR experiment. Remember to include a couple of non-differential genes as controls, though.

Got it! Thank you HESmith.