HI, there isn't ONE housekeeping gene that fits for all experiments. Try different ones and then you will see which one will be the best for your own experiment! Cheers, Nadine

HI, there isn't ONE housekeeping gene that fits for all experiments. Try different ones and then you will see which one will be the best for your own experiment! Cheers, Nadine

The best "one"?

Please be aware of the MIQE criteria:

Bustin et al: http://www.ncbi.nlm.nih.gov/pubmed/19246619

You can use three HK genes like b-actin, HPRT and a-tubulin. This should give you more confidence for normalization, especially if they behave the same way from sample to sample

I'm dubious about any PCR using only one single HK-gene for normalization. Studies show, that different tissue have different HKs suitable for PCR normalization. In our lab we tend to use triple control (beta-actin, GAPDH, H6PD). These are mostly universal, still, I'm sure one can find and exception even for these 3. good luck!

In our hands we have found that B-Actin has been a good housekeeping gene for working with fibroblast like synoviocytes. It remains fairly stable during inflammatory stimulus (at least with the stimuli and concentrations that we are using). The best thing is to test a couple using your conditions and see what works best.

Good luck

Http://www.tataa.com/products-page/gene_expression_assays_panels/reference-gene-panel/

My personal experience is that Beta-actin works great. You can although keep along GAPDH, just to be sure. As a dry run, may be an Agarose gel following reverse transcription with a few equimolar samples and the constitutive genes can tell you if it is working fine, if you get same intensity bands with all your samples.

I have used GAPDH the most, but as mentioned above, using multiple reference genes is much better. Your choice should be a gene that is very highly expressed so that small variations in expression between samples do not have a detectable effect on overall expression. Further, if you use more than 1, they should have different functions. That is, GAPDH is utilized in metabolism. Therefore, your other choice should have a different function (like cytoskeleton - actin, tubulin; or mRNA translation - like 18s). If you choose two reference genes that have similar functions, like actin and tubulin, then conditions that affect one of your reference genes are more likely to affect your second. If they have different functions, then it is less likely they will both be affected by any one condition, making your control more stable. Lastly, you should ensure that your reference is not affected by your treatment. This can be difficult, because you don't have a reference gene for the reference gene you are testing. But you can examine the literature to see if there might be any effect and do test reactions with well quantified starting mRNA/cDNA to look to see if there are large differences. Good luck!

Here is a good reference for picking and normalizing multiple reference genes:

Vandesompele et al., 2002 - Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

i prefer to use RNA polymerase II, GAPDH and HPRT, i found RNA pol is stable and is supposed to increase/decrease in response to demand. If you use more than one then use the geometric mean of all your housekeeping genes as the input Ct and you should overcome the variation. be wary of B-actin, it will always work which is why so many people use it but it is really variable.

I think Beat-actin will work for you for this type of tissue, although you can try GAPDH also .......

I found GAPDH variable and unreliable, HPRT and 18S are good to try out.....it all depends on your tissue. There is a PCR array from SABiosciences that will tell u what HKG is best for your tissue or cells.

Recent investigations show that the correct way is to try 5 different houskeeping gene and with Gene Norm program analyse the result and determine the most appropriate houskeeping gene for your experiments. If you have not these posibility I suggest using beta-actin. Do not use GAPDH, it is too variable expression and can mask the results. I have personal experience!

Hi, As Nadine mentioned, there are quite a number of housekeeping genes. But, I personaly have the experience of using GAPDH and beta-Actin for almost the same experiment. Good luck.

I have used Beta-actin, GAPDH or the rpsa ribosomal subunit with good results ranging from a variety of different cell lines to primary cultures to tissues..

Hi!

I usually use 2-4 house keeping genes (RPL30, RPL27, B-Actin, GAPDH etc). The more the better (it works well with REST software from Qiagen as well: Pfaffl, M.W., Horgan, G.W.& Dempfle, L. Relative expression software tool (REST) for group-­‐wise comparison and statistical analysis of relative expression results in real-­‐time PCR. Nucleic Acids Res. 30, e36. (2002).

Please check also: de Jonge HJ, Fehrmann RS, de Bont ES,

Hofstra RM, Gerbens F, Kamps WA, de Vries EG, van der Zee AG, te Meerman GJ, ter Elst A: Evidence based selection of housekeeping genes. PloS one 2007;2:e898.)

Good luck!

what about 18S?

Hi Daniel,

I agree with all the comments above. It is difficult to suggest a particular HKG for a particular study, as there is no gold standard but I strongly recommend to read the article i.e. Evidence based selection of HKG which Biljana has suggested (above) it will give you some insight into how a proper HKG should be chosen. Always go for more than one HKG and if you can stay away from GAPDH is better. I am currently working with prostate cancer cell lines and tissues and normalizing my data to TBP, 18sRNA and GAPDH, so happy with TBP and 18sRNA but not GAPDH so far! It seems that tissue conditions are really affecting the expression level of GAPDH !

Good luck.

The “ideal” reference gene should be transcribed in all cell types and tissues under study and transcription should be constant relative to global cellular transcription rates under different experimental conditions. Expression of individual reference genes does differ among samples under different experimental conditions. Historically, the most commonly used reference genes were β-actin and GAPDH, as they were used for many years in Northern blots, RNase protection assays, and later in conventional RT-PCR. However, recent studies have indicated that these common reference genes are not stably expressed under all experimental conditions.

I suggest you should validate 5 to 9 difference HKG to find which HKG should be chosed under your experimettal conditions.

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As above there is no single house keeping you have to simplify your work. Either you could try a couple and narrow down on one that ia least deferentially regulated. For such an experiment we accurately quantify RNA (control and treated or normal disease) using either a dye based (e.g. Qbit) or the bioanalyser and then use that cDNA to determine the best HKG. Alternately you could use data from microarray data sets that have been previously published (if done for your tissue condition) and shortlist from that and use.

Finally in my experience on a variety of tissues, 18S works best for most (only one developmental study we found it varied), beta actin is highly variable, mainly because it is modulated by steroids and in inflammation.

If you need accurate results DO NOT go by the standard assumption that because majority of researchers have used X HKG so it can be used. It can be misleading and bias the whole study (and I have had experience on that).

Best luck

Deepak

hi every one..........there is no exactly house keeping gene can be used,instead that you can try more than one until reach to the best one.however the comparison of house keeping gene depend on the gene itself expression due its expression is the same in each condition with there feature is small gene..I think many house keeping gene like GPDH,Beta actin, 18rs, B2M and other

Call them "Reference Gene" , not Hosekeeping. Since not all housekeeping genes are valid as reference, you should avoid the term and use "reference gene" instead

I would like to suggest you 26S and GAPDH which are used for many experiments for normalising.

I have previously used beta actin/GAPDH/cyclosporine depending on the cell lines

have u never tried 18S?

Dear Daniel,

I would recommend you to have a look at the most stable genes in your tissue under similar experimental conditions in Refgenes (http://www.refgenes.org/rg/) it is free and very helpful. More reading:

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics 2011, 12:156.

Good luck with your experiment.

http://www.refgenes.org/rg/doc/GV_refgenes.pdf

I completely agree with Nadine. You should try at least 6-7 housekeeping genes and look which works for you better. As it is said there are no perfect house keeping genes which will fit all your experiments. For us after trying a panel of housekeeping genes, 18s and ACTB worked fine. I would recommend you to go through the below link. It will help you standardize your desired housekeeping gene.

http://www.ncbi.nlm.nih.gov/pubmed/12184808

The group consensus is correct. Its best to try a couple like GAPDH or ß-actin. We have even used ß-III tubulin for some cells type/tissues. We frequently use 18S for our straight qPCR for genome number controls as it is more consistent between different cell type/tissues. have not looked at it in expression studies however.