I am preforming RT-qPCR validation of novel identified miRNAs; however, I'm having difficulties with dilutions of my sample, because higher dilutions (for example 10000 fold dilution) have lower Ct (for example Ct 20), whereas lower dilutions (example non-diluted or 10 fold diluted) have higher Ct (example 23 or 24). Is this an inhibition or what is going on in my RT or qPCR reaction? I’m performing stem-loop RT and SYBR Green qPCR.
Thanks for helping!
Hi.... first using SYBR Green qPCR is not good technique... read the attached article...it can be helpful for you...., Good luck
It is an odd situation. My main suggestion is to run a NTC for the RT-qPCR if you haven't already done one. Does the NTC amplify at a relatively low Cq? Also do a melt curve after your amplification to check it and compare between the serial diluted samples and NTC.
Thank you both for your answers.
I always run a NTC (water) and there is no amplification present above the threshold. And regarding melting curves, between serial dilutions, there is no difference in melt curve (I also always perform melt curve analysis), I observe only one distinct peak at the same Tm for all dilutions, or sometimes a little difference between technical replicates. But nothing alarming regarding melt curve.
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