need help for trouble shooting the contamination in miR-X assay from serum - (Jun/06/2013 )
I am trying to find out the presence of a specific miRNA: miR-X in the serum of patients. I was getting good results in the beginning, when I was doing a relative gene expression using U6 an a endogenous control. But later I came to know that U6 is not a reliable control for serum miRNA profiling.
Next I have now tried to spikein non-homologous cel-miR-39 . And also I have started using synthetic RNA oligonucleotide for the generation of a std surve. The std curve is running smooth, however I have been doing the target miRNA std cDNA prep in the same room where I have isolated miRNA. And now I am ending up getting amplification for the target miRNA even in water samples. All the healthy controls are positive for the target miRNA-X. I am stuckup and find no way to rule out wat has exactly happened.
Somebody please help me solving this problem...
somebody please try to help me in finding out a solution to the above problem i have described