I have used mircury exosome serum/plasma kit along with trizol for extraction of my miRNAs from human plasma. RNA concentration was 623.8ng on nanodrop with 260/280 ratio of 2.12. For RT-PCR and qPCR, I have used mircury reagents and LNA primers.The cDNA dilution was 1:9, 1:8 and 1:12. On agarose gel electrophoresis, band was visible only for spike-in (UniSp6). Kindly find the images attached for reference.
Thank you!
What's most likely going on is that your gene of interest is not being amplified at all and you are just seeing the baseline fluorescence in the tubes. Did you include a positive control sample in your qPCR?
Hello
Yes, i agree with Katie A Burnette; your interest gene is not being amplified .
Good Luck
Thank you for the response.
No, I did not have any positive control for the target gene but I did put a spike-in which was amplified. Should I modify the extraction protocol in this case?
Dear Shalini
Check the purity of your sample using nanodrop with absorbance on A260 and A280
Regards
You should always include a full set of controls for qPCR (positives, negative, no template, etc.)
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