Dear All,
In my western blotting experiment I found a decrease level of both Akt and pAkt expression. So I want to check the mRNA level of them. I am wondering which primer should I buy for the RTpcr. Thank you very much in advance.
Best Regards,
Jun
I think you can measure mRNA levels of total Akt only with RT-PCR. You can do IHC or ELISA to check activity of Akt.
I'll try to help you but you must answer the following questions:
1. Which dye will you use, sybrgreen or taqman probe?
2. What are the genebank numbers of genes?
3. Are you going to use housekeeping gene? and which housekeeping gene?
The following article can help you.
Active form of AKT controls cell proliferation and response to apoptosis in hepatocellular carcinoma
Oncol Rep. 2014 Feb; 31(2): 573–580.
Published online 2013 Dec 16. doi: 10.3892/or.2013.2932
You can use the following primers
GAPDH Tm 62
Forward 5'-GGC TGA GAA CGG GAA GCT TGT CAT-3'
Revers 5'-CAG CCT TCT CCA TGG TGG TGA AGA-3'
AKT1 AKT serine/threonine kinase 1 [ Homo sapiens (human) ] Tm 57
GeneBank No: NM_001014431.1
Forward 5'-ACG TGG TGT ACC GGG ACC TCA A-3'
Revers 5'-AGG TCT TCA TGG TGG CAC CGT C-3'
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