Hello everyone ! I am using mirVana miRNA isolation kit for the isolation of total RNA containing small RNA. Further i used Agilent first strand synthesis kit(cat#600036) for the synthesis of mi-cDNA. For the primer designing , i used the sequence of my mir of interest (miR-144-3p) as the forward primer. However, I am not able to get any amplification using SYBR Green qPCR method. I am using PowerUp Sybr Green Master mix for qPCR. Kindly suggest what is the underlying problem for the amplification failure?
Yes , i have run my cdna samples for U6. And the ct value for all my mi-cDNA samples is in the range 17-21. So, the reverse transcription is successful .
i am facing the same problem in miRNA. I am using SYBER II also.. I asked the Primer Company.. they recommend to use TE or RNase free water to dilute the Primer as its RNA sequence and easily be degraded by RNAases.. I will try that tomarrow.. my SNORD95 and U6 is Ok.. see Picture bellow.. others m4 miRNA in all the samples is zero..
Hi
I also faced same problem with cell ro ct kit I was getting proper apmlification but mirnasy it was not proper. also came across paper saying triozol or quizol also will have preferential solubility bias towards mirna.hope that answers
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