Dear Mohd,
I would think that tubulin and actin are often standard controls in real time PCR as they are abundant so that ensures good readings. Importantly, when choosing a housekeeping gene, the most important think is to ensure that the expression of this particular gene does not vary in your experimental conditions (i.e. drug treatment, knockout, silencing...). In that sense, I guess that those genes are quite stable. However, this is not a universal fact, it is very important to test the expression of few housekeeping genes, at least four I would say, with the treatments or experimental conditions that you are gonna use to validate that there is not fluctuations of the gene expression.
Hope this helps!
Hi Mohd
- agree with Laura: normalising genes are included to correct for fluctuation between cDNA samples that have nothing to do with intrinsic variation in expression or cDNA copy number
- cDNA samples derived from 2 samples of RNA might vary by virtue of the quality of that RNA and hence the efficiency of RT which will have an impact on how efficiently your subsequent PCR occurs and hence Ct value. This has nothing to do what so ever with the effect of treatment on copy number (expression)
- In addition impurities for example present in RNA that effect RT efficiency and hence cDNA copy number might even affect the efficiency of down stream PCR
- Finally normalisation will correct for variation in apparent Ct values and thus expression linked to variation in the amount of components added to the reaction in turn connected to pipe tying errors
- By eliminating these confounding factors normalised data - that is the difference in Ct value between your (GOI with treatment - housekeeper with treatment ) - ( GOI control - housekeeper control) represents a true difference in expression between these 2 sample types ( biological replicates )
- Regarding what housekeeper to use as explained by Laura this is dependent on biological context; that is tissue and treatment type. The crucial thing is that treatment does not affect expression in your tissue and that the housekeeper you have chosen is expressed at a high stable level in your tissue.
- Spliceforms of particular housekeeping genes can vary between tissue types in a particular species so when designing primers/probes ensure they are pan specific and crucially only amplify the same set of isoforms of your housekeeper
- certain genes like gapdh and tubulin tend to be used a lot but in truth as mentioned by Laura and based on the above considerations you ideally need to evaluate at least 5 housekeepers to select the optimal 2 for your assay context
- Gene Norm Norm finder or ref finder are typically used for selecting the best housekeepers:
- http://fulxie.0fees.us/?i=1
- https://genorm.cmgg.be
- http://moma.dk/normfinder-software
- Alterbatively look for precedents in the literature for your assay type
Dear Mohd Ikmal Asmuni
Pls. find the attached files
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3825189/
https://bmcplantbiol.biomedcentral.com/articles/10.1186/1471-2229-8-131
http://link.springer.com/article/10.1007/s00438-010-0511-1
https://presans.com/sofia/expert/232201091/youjun-zhang
http://www.gene-quantification.de/hkg.html
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097469
https://academic.oup.com/jxb/article/56/421/2907/593469/Housekeeping-gene-selection-for-real-time-RT-PCR
http://bmcplantbiol.biomedcentral.com/articles/10.1186/1471-2229-8-131
http://bmcmolbiol.biomedcentral.com/articles/10.1186/1471-2199-10-11
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000100019
https://bmcplantbiol.biomedcentral.com/articles/10.1186/1471-2229-6-27
Hoping this will be helpful
Regards
Prof. Houda Kawas
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