qRT-PCR protocol for an extremely tight budget

[RatRace]

Hello,

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.

PMID:
12753500

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

[admin]

Hi Andrew welcome to the site. I am sure someone can help you and I have done many PCRs without buying kits however it takes some optimization. Have you checked your old kit to see what is in their mix? You could buy all the components separately and prepare a similar mixture.

[NATHANALANPATRICKROGERS]

Due to the design of your experiment and new cost considerations, there may be no need to outlay extra for qRT-PCR technology. You could achieve the needed degree of discrimination by using a commercial RNA extraction kit, GeneChip to confirm gene followed by a straightforward qPCR using your selected primers.

[RatRace]

Originally Posted by admin

Hi Andrew welcome to the site. I am sure someone can help you and I have done many PCRs without buying kits however it takes some optimization. Have you checked your old kit to see what is in their mix? You could buy all the components separately and prepare a similar mixture.

By "many PCRs" are you referring to qRT-PCR? We have done PCR amplification without a kit; we order the polymerase, dNTPs, primers, etc. as needed. As far as qRT-PCR, we haven't done that without a kit.

[RatRace]

Originally Posted by NATHANALANPATRICKROGERS

Due to the design of your experiment and new cost considerations, there may be no need to outlay extra for qRT-PCR technology. You could achieve the needed degree of discrimination by using a commercial RNA extraction kit, GeneChip to confirm gene followed by a straightforward qPCR using your selected primers.

Are you suggesting we use a custom gene array or something? I don't think using another RAE230 GeneChip would help.

qPCR and qRT-PCR are synonymous AFAIK.

[NATHANALANPATRICKROGERS]

Originally Posted by RatRace

Are you suggesting we use a custom gene array or something? I don't think using another RAE230 GeneChip would help.

qPCR and qRT-PCR are synonymous AFAIK.

I think standard PCR has been used to quantify gene expression for many years but it has recently been overshadowed by RT-PCR. Standard PCR will be cheaper and less accurate; however if you do manage to buy cheap RT-PCR components that work then well done. The choice depends on the type of research being pursued and the quality of results soughtafter.

[RatRace]

Originally Posted by NATHANALANPATRICKROGERS

I think standard PCR has been used to quantify gene expression for many years but it has recently been overshadowed by RT-PCR. Standard PCR will be cheaper and less accurate; however if you do manage to buy cheap RT-PCR components that work then well done. The choice depends on the type of research being pursued and the quality of results sought after.

We're looking for results that are accurate enough to measure fold changes in expression.

[RatRace]

For those who are interested, someone was kind enough to point me to a great reference on homemade SYBR-Green qPCR mixes:

Pellissier F, Glogowski CM, Heinemann SF, Ballivet M, Ossipow V. Lab assembly of a low-cost, robust SYBR green buffer system for quantitative real-time polymerase chain reaction. Anal Biochem. 2006 Mar 15;350(2):310-2.

PMID: 16434019

[sojajos]

So did you try it? What is the cost?