We did Affymetrix RAE230 GeneChip analysis of Dahl rat RNA from three strains and three diets (high salt, elevated salt, and low salt). We need really good quantification to confirm our genes that we identified as differentially expressed.
We have a MyiQ Single-Color Real-Time PCR Detection System from Bio-Rad that uses SYBR-Green I. We did qRT-PCR before with kits and our qRT-PCR results totally conflicted with our GeneChip data for the differentially expressed genes. However, we designed primers for the entire transcripts, instead of amplifying the specific subsequences that the Affymetrix probesets were targeting.
Anyways, we lost all of our funding and we are on an extremely lean budget. The PI found an article about making an inexpensive PCR kit from component parts.
The reference is:
Ramos, P et al. "Quantification of Cytokine Gene Expression Using an Economical Real-Time Polymerase Chain Reaction Method Based on SYBR Green I." Scand J Immunol. 2003 May;57(5):439-45.
The fulltext is available free. Just type the PMID into pubmed and there's a link. I would post the link, but I don't have enough posts to do that yet.
So does anyone know anything about making qRT-PCR mixes without buying a kit? We would really, really appreciate any help someone could offer.
Andrew McSweeny, MS
University of Toledo College of Medicine