I want to made a pair of primers of GAPDH for the QRT PCR assay.
There are six RNA variants to GAPDH in the NCBI , these variants code to three protein isoform, the variant 1, 3 and 4 have the same isoform, the isoform 1, the variant 2 code to a shorter isoform of the isoform 1, the variant 6 is a non code variant, and the variant 7 code to isoform 4.
Does these variants interfere?
I can made a pair of primers whitout the 6 variant of RNA, but i cant do primers more specific than that.
how can everyone use the gene GAPDH like reference if has all these variants and enhancers, promoters and variables?
Assuming you are using a human/mouse model, are you also aware of the the pseudogene issues with GAPDH?
" A quick glance at figure S5 immediately leads us to the fact that there are so many processed PGs of the mouse Gapdh which are 100% or almost 100% identical to the Gapdh mRNA"
Article Pseudogenes as Weaknesses of ACTB (Actb) and GAPDH (Gapdh) U...
Please, be carefull with GAPDH, for me: choose another feference genes. It could be much better. For example in colorectal cancer tissue samples you can use SCARNA5 - it is quite stable in fresh frozen tissue. May be RNU1 and RNU12 in the case of breast cancer (her-2 negative). I always conduct analisys of candidate reference genes before starting use as a reference. Moreover in the case of normal and tumor tissue expression stability can vary.
- Hi there
- Yes you are right
- It is important to take accoutn of different isoforms for qRT PCR
- Thus, when I design primers to GapDH I will design them to an exon or better still over an exon-exon boundary (making them specific for mRNA) in a junction or exon that is found in ALL KNOWN splice variants
- Thus, you would not run the risk of expression being skewed by variable or absence of expression of a particualr variant (isoforms tends to be expressed in a tissue specific manner)
- Apart from using a common exon/Junction, I would also recommend using the last (most 3') common exon/junction you can find to maximise efficincy of cDNA production
- Thus, if all isoforms share a region of the 3' UTR then place you primers in the 3' UTR
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