I'm using 18SrRNA and β-Actin as housekeeping genes in my qPCR experiments. GAPDH and HPRT1 have higher Ct. However in many samples (CTCs), the 18SrRNA seems to be more accurate.
Selection of reference genes is a delicate issue.
For each tissue/cell type, activation/stimulation/disease stage etc. one has to find the RG that is most stable between conditions.
Gapdh and ß-Actin primers have to be selected very carefully to exclude psudogene amplification in case there is gDNA contamination in RNA samples. You would be much safer with 18s rRNA, ß2m and HPRT.
Note this tip which may help to solve your problem. Real-time RT-PCR-specific errors in the quantification of mRNA transcripts are easily compounded with any variation in the amount of starting material between the samples, e.g. caused by sample-to-sample variation, variation in RNA integrity, RT efficiency differences and cDNA sample loading variation (Stahlberg 2003 2004a 2004b). This is especially relevant when the samples have been obtained from different individuals, different tissues and different time courses, and will result in the misinterpretation of the derived expression profile of the target genes. Therefore, normalisation of target gene expression levels must be performed to compensate intra- and inter-kinetic RT-PCR variations (sample-to-sample and run-to-run variations) (Pfaffl & Hageleit 2001). You included b-actin, GAPDH and HPRT1 of which GAPDH and HPRT1 are reported to show variation in some tissue. See reference GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues by Barber et al Physiol Genomics 21: 389–395, 2005.
However, in most of the papers, they use GAPDH. I've tried 18SrRNA in more than 50 samples, in different conditions of culturing cells and seems to be the most reliable. The 18SrRNA is also expressed after mRNA isolation (beads). In this case, should I use it as a housekeeping gene? Because the gene expression profile of 18SrRNA and βActin is different.
In principle, before starting an experiment you have to check if your house keeping gene is influenced by stimulation factors with which you're working. When I was going for some gene expression analysis in a CML cell line to find out working mechanism of two specific drugs using Affmetrix gene array, I could not use GAPDH in confirming real-time PCRs due to the fact that I have siginificant changes in GAPDH gene expression after treatment of cells compared to control cells. Same can happen with beta-Actin or any other gene as each and every gene has some function(s) and you have to check first if they're influenced or not and if they are suitable as house keeping gene under your conditions or not. To the best of my knowledge, 18S rRNA should be very stable and therefore be a good target as house keeping gene.
If you have access to microarray data that reflect your experimental conditions/treatments you can check overall signal intensities for your genes of interest and your prefered reference genes. Based on that you pick 2-3 RGs that match the signal intensity/expression level of your genes of interest.
Then check the stability of those reference genes with the tools available - Normfinder or similar.
Especially when working with genes that have very different levels of expression - let's say imaginary expression values of 30, 200 and 9000 - it is handy to use RGs that mirror these changes.
The cell lines I'm working are cancer cell lines, or commercial or patients' derived (from CTCs). When I isolate total RNA (with TRIZOL or RNeasy) I always use the 18SrRNA as RG. However my problem arises when I started to isolate mRNA from these cell lines. Thank you all for your suggestions/informations.
As I have mentioned above - I was going for Affimetrix gene array as well as real-time PCR for confirmation of gene array data. I was doing this on primary CD34+ hematopoietic stem cells from CML patients as well a cell line. In both cases - gene arrays as well as real-time PCR we finally work with mRNA, so this should be the same condition you have, the only difference will be that you have different cell lines.
And then it is according to your specific question normally you will treat cell under different conditions - and the basic question is whether these conditions have influence on your house keeping gene or not. If you choose e.g. GAPDH but want to investigate glycolysis, this would be a bad choice as GAPDH will definitely change its expression. Same is with beta-Actin when you want to investigate changes in the cytoskeleton. Have in mind, every gene has some role in the system - and only if nothing related to the specific role / function of the gene it will be suitable.
I was working with the software Normfinder after going for gene array, I one condition it was working good and giving a nice reference gene, in another condition it was not working as I always got a delta Ct value of 2 meaning that the "reference gene" was 4-fold differentially expressed. Therefore, I finally took some 18S rRNA primers also for my real-time PCR and got a stable reference gene.
Hi, I would like to ask how much of variation in the Ct values of the HKG among experiment and control samples is accepted. For examples HPRT gene Ct values for the control group varies from 31 to 34.7 (median 34) whereas experiment group (genotype difference for ex.) is ranging from 30.6 to 34.8 (median 31). The analysis is carried by using deltadeltaCt method, which is widely accepted and reliable. As quite similar Ct values are expected from HKGs, I am not sure of the variance in my dataset. I would appreciate if anyone can comment on this. Thanks.
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