DNA copy number-QPCR - (Mar/30/2005 )
Hi,
when I work with DNA to detect copy number changes, what kind of reference should I use? Shall I use GAPDH or a different gene which shows normal in the whole genome?
Is there any difference to design primer for DNA and cDNA? I mean for cDNA, my primers were located in two different exons. How will I design primer for DNA? Thank you very much.
Dear molcyt,
Are you working with gene expression?
Regards
yong
Dear Yong,
No, I am working with copy number change detection (amplification, deletion). For this reason I have to work with DNA.
Hi,
You cannot use cDNA based designed primers for doing qPCR, the reason being the amplicon (from the genomic DNA )likely exceeds the maximun allowed (150 bp) for real-time PCR. If you are using tumors, select a reference gene from a chromosome known to be least affected in the specific tumor you are studying. You may try GAPDH, albumin, beta 2 microglobulin, etc. If you wish, you can design a reference from the above mentioned genes using the corresponding BAC DNA sequences. Try to use an intron while designing. Make sure, there are no SNPs! Hope that helps.
SV
Dear SVTX,
Thanks a lot for that information.
You cannot use cDNA based designed primers for doing qPCR, the reason being the amplicon (from the genomic DNA )likely exceeds the maximun allowed (150 bp) for real-time PCR. If you are using tumors, select a reference gene from a chromosome known to be least affected in the specific tumor you are studying. You may try GAPDH, albumin, beta 2 microglobulin, etc. If you wish, you can design a reference from the above mentioned genes using the corresponding BAC DNA sequences. Try to use an intron while designing. Make sure, there are no SNPs! Hope that helps.
SV