As usual, the community was extremely responsive in my query to using housekeeping genes for RT-PCR. Since so many people wrote to me asking for a summary of the responses, I present here slightly edited versions of most of the EMails. Some of these were already sent to the Cytometry list, but I thought it would be useful to list them all. I chose to remove personal contact information in case some of the people providing advice preferred anonymity by default; if anyone would like to get in touch with a specific contributor (most of whom offered to provide more detailed information upon request), I can EMail your request on to the appropriate person. Again, thanks to everyone for their advice, especially on this slightly off-topic query. mr >In the last experimental design evaluating TAP1/2 mRNA (TAC-MAN) and nef- vs. >nef+ gene constructs it appeared that transferrin or GAPDH mRNA was >superior to >beta-actin. This was measured on PBMC's stimuated with PHA/IL2 over >14 days and >infected over 6 days. I know the GAPDH does take advantage of the splice >juction but I am not sure about the others. >I have used G3PDH (glyceraldehyde 3-phosphate dehydrogenase) for semi >quantitiave PCR using RNA. The gene contains introns so you can design >primers that overlap the junction in order to amplify RNA products and not >genomic DNA. This worked very well for the system that I was using at the >time (cytokine receptor transcripts in stimulated PBMCs). I have used it as >a control in several PBMC populations (T cells, B cells, macrophages and >neutrophils) and have not seen any amplification even using strong mitogens >such as PMA/ionomycin. >we use GAPDH and L32 for our RNAse protection assay. >We have found glycerol-3-phosphate dehydrogenase to be a more suitable >housekeeping gene that beta-actin for quantitative RT-PCR. It is >interesting that the primers we use amplify cDNA from a number of >different species. I am pretty sure our primers do take advantage of >spanning an intron or a splice junction as you worded it. We have found >similar levels of expression on an cellular basis regardless of whether >the cells are unstimulated in culture or stimulated with mitogens or >other stimuli. Of course, this was also true of beta-actin, but not >quite as consistent. >We are using the GAPDH >housekeeping gene and DO take advantage of an intron to be able to >distinguish genomic DNA and cDNA PCR amplification products. It is my >understanding that the sequences and primers were originally designed by >Francois Villinger. They're based on the human GAPDH gene (but are >conserved in rhesus). They are also using a different GAPDH primer >set for Taqman amplification and we're currently developing more Taqman >probes for a number of genes to follow during SIV infection (including >viral RNA/DNA) (no other housekeeping genes as far as I know). Hope this >helps. >Try looking at 18S or Gus, ABI sell Taqman Endogenous Control using the >7700 Taqman gene expression technology. Look at the ABI site, PEbio.com >under PCR -PDAR's >RT-PCR is performed with the Superscript One-Step RT-PCR kit according to >the instructions of the manufacturer (GibcoBRL). In a first reaction tube, >the total RNA is "titrated" by measuring the amount of 28S rRNA (ribosomal >RNA) with primers specific for the 28S rRNA after addition of a known >amount of an internal standard RNA which is co-amplified with the cellular >28S rRNA but yields a different (shorter) amplification product. This >internal standard is a custom construct obtained through a local company >called SwissClone. The RT-PCR products are then separated on an agarose >gel, stained with ethidium bromide and a digital picture is obtained with a >Kodak DC-40 camera. The intensity of the two bands (28S rRNA and internal >standard) is measured on the digital picture file using Kodak Digital >Science 1D software and the R1 ratio calculated as follow: R1 = intensity >of the 28S RNA signal / intensity of the internal standard signal. >In a second tube, the IL-1ß mRNA (for example) is amplified by RT-PCR >together with a known amount of internal standard (pAW109 RNA from Perkin >Elmer in this case) which is added in the same tube as internal standard. >Again, two distinct amplification products are obtained, separated on an >agarose gel, quantified using the Kodak system and the R2 ratio calculated: >R2 = intensity of the IL-1ß signal / intensity of the pAW109 signal. >The relative IL-1ß mRNA expression is then deduced by computing R2 / R1. >The results are expressed in % of the negative control expression. > The PDH beta subunit (Accession No >D90086) is a good one. The mRNA of this gene is constitutively express in >lymphocytes and suffers minor changes after activativation. I have used >primers: GTTTGGCAGCGGATAGAGGACACG and CATTCTCTAGCACCACCACTGGAT that I >designed myself with the aid of the DNA Star program. The amplicon size is >637 bp (spanning at least one intron) >At the moment we are amplifying the abl message and found the information >present in "Debate Round-table: Multiplex PCR for quality control of >template RNA/cDNA in RT-PCR assays. F Watzinger and T Lion, Leukemia year >1998, page 1984-1986 (can't find the volume reference)" very useful: these >people report on the use of bcr, abl, b2-MG and PBGD (porphobilinogen >deaminase) in a multiplex assay! >We are using the ABI Prism 7700 Taqman system for our QPCR. This assures >more reliable quantification and reduced workload compared to classic >competitive PCR methods (which we still use for some targets). The demands >that we make for housekeeping genes are the following: >- the expression level should be similar between the control gene and the >target gene >- expression of the control gene is not effected by therapy, infections, >etc... >Besides this we try to avoid genes for which many pseudogenes are known >(such as beta-actin) and we always place our primers over an exon junction. >I use GAPDH, but have primer designs for pyruvate dehydrogenase and histone >3A. One potential problem in GAPDH is the presence of pseudogenes although I >have not personally experienced this problem. >We do not have a 7700 so one work-around we have used with partial success >has been the Ambion competimer strategy. About equivalent to our success >with competitive PCR (not good enough). However that took considerable >fiddling to get it right (which Ambion blamed on the rarity of our target >message) >Beta 2 microglobulin was recently suggested to me on the basis that ALL >cells express it (even my meso/epithelial cells where it too is a rare >message). >GAPDH is my choice for a good housekeeping gene. Yes, I always try to use >primers that span an intron (or a few) if I can. Also, you could run a PCR >reaction with RNA only to see what level of genomic DNA contamination you >might have. >in terms of housekeeping gene controls, for mouse gapdh is >pretty standard and in human b2 micro globulin (i'm pretty sure, it's >been a while). someone's email response mentioned beta actin, we've >found that it is more highly regulated than gapdh or b2m. all exon >probes. for the determination of gene regulation as well as specific >cellular gene expression you have to use different cells, types and >lines, under different but morphologically, phenotypically, or >genetically defined conditions. >I have just finished "troubleshooting" housekeeping gene primers for >quantitative real time RT-PCR. After some fairly extensive work, I found >HPRT to be the best. Both beta actin and GAPDH (a.k.a. G3PDH) amplify >genomic DNA.
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