I have isolated RNA from 50ul serum and eluted the RNA in 30ul of DEPC- treated water. But when i am preparing 10ul of cDNA from 1ul of the RNA and using it for amplification of a 115bp established marker product, i get a blank gel.
Hi Subham,
First of all, make sure the used primers are well designed and specific.
Secondly, I highly recommend to measure the A260/230 to determine the conc. of the RNA and to exclude the presence of contaminants which could adversely affect the process cDNA synthesis.
Best wishes,
Radhwan
Hi,
in addition the detectability also correlates with the expression level. So a strong expressed transcript will be easier detectable than a low abundant target. Do you have a rough idea about expression levels? Perhaps you could use a high abundant sequence for optimization of the system (in case this is not already true for your used system)
Sven
I would check to make sure your RNA isn't degraded for whatever reason, assuming that you ahve enough RNA in that 1uL...
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