Hii
I have reprogrammed a cancer cell line with conventional four factors USING LENTIVIRUS-POLYCISTRONIC OCT,SOX2,KLF4,cMYC. Now I GOTspheroidss after days of INFECTION. Spheriods are expressing alkaline phosphatase nicely. expressing surface markers, ssea4,tra-1-81,tra-1-60. But not positive for endogenous gene expression of oct4 and nanog in RT PCR. How should i go forward from here to get an endogenous expression of these OCT4 and NANOG.
for Reverse transcriptase PCR. I have isolated RNA from spheroids plated in one 12 well plate. the yield was 1micro gram per micro litre. 260/280-2.1
house keeping gene beta actin is positive from cDNA of above RNA, but not OCT4 and NANOG.
can i try small molecules to make it positive for these genes. if so what small molecule how much concentration and how many days.
what i should do to the spheriods to make it positive for endgenous oct4 and nanog.
Please help
Dear Bindhya,
You need to do two things;
1. standardize your reprogramming
2 standardize your molecular biology expt
Assuming you got iPSCs, you need to standardize your RT PCR. When you do RT-PCR include a positve control, take RNA from iPSC cells or you can buy commercially available RNA or from some research lab which does iPSc work. If your primers work with positive control, then your be confident about OCT4 expression in your iPSCs.Make sure you are taking 1-2 ug RNA for cDNA synthesis.
Hope this helps.
Regards,
Prasad.
Dear Prasad
Thanks for your reply.
I had a positive control to make sure that my primers are working.
Dear Bindhya,
If you had reference RNA from iPSCs and that showed OCT4 NANOG, but not your spheroids, then it raises questions about the SSEA-4 and TRA staining. Cancer cells have elevated endogenous AP activity. I do not know whether your reprogramming has worked in this case.
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