I have reconstituted the RNA from RNA stable tube biomatrica catalogue number 93221001.The 260/280 was 1.81 while 260/230 was 2.The ct value for GAPDH is 27 .Will this value is acceptable.What would be the probable cause of such high ct value.Source of RNA was human peripheral blood.The same ct value was obtained from RNA which was freshly isolated and not stored in RNA stable kit.The 260/280 was 1.83 while 260/230 was 1.73.
Kindly help me for troubleshooting
@ Wei-Kang Lee , I am also worried for my gene of interest , although I have used 1 ug of RNA
NO ,I have done only 260/280 ratio and yes I have converted RNA into cDNA
Unless GAPDH is expressed at low levels in your tissue, I also think that your gene of interest will have high Cts. Can you do a test run to gauge its (GOI) expression? I am suprized that given the 1 ug RNA input you are getting moderate abundance for GAPDH (but I don't know if this is the norm for peripheral blood). Maybe check your calculations for cDNA input and primer re-suspension. I have had a similar issue before with late Cts and my mistake was in the primer re-suspension which had me using a lot less primer than what I should've been using!
As Wei-Kang recommend, you should also check the RNA integrity of the blood sample.
Parul you can try 18srna as control as sometimes you may get lower value for GAPDH and try using some high efficiency cdna kit synthesis in order to get correct value for GAPDH so that you can get clear idea about your gene of interest.
Thanks Chandni , do you have human 18srna primer , I am using cDNA kit from biorad. Which kit you used ?
Wei-Kang Lee :Thanks for adding your answer , yes I agreed , there could be a possiblity of degradation.
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