I used Qiagen miRNEasy micro kit, miScript RT II kit and miScript SYBR greenPCR kit with Qiagen custon primers assays for mir-499 and mir-208. No-matrix PCR showed one-peak melting curve and the C(t) was 30, when in positive control C (t) was 34. I cheсked twice, so this is not because I changed the tubes. How it is possible?
Greetings,
First of all, are you using the recommended PCR conditions with these primers. Secondly, amplification at such high count of cycles are mostly due to primer dimers or small amounts of crossover contamination, as they are around 100 or less copies. However, there's a small chance of the presence of inhibitors on your reaction, wich could be fairly assesed by evaluating the quaity of the geneic material you are using as raw material. I'd double check the used PCR conditions and or reextract - synthesize cDNA to be sure this kind of issues is not due to contamination of my stock solutions.
Good luck
Adán Valenzuela Castillo, thank you, I'll try to reextract RNA and make new cDNA
Hi Kristina,
you can check the amplification by in vitro PCR and see if primers are well designed (http://genome.ucsc.edu/cgi-bin/hgPcr or https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). Can your primers amplify something by self hybridization?
fred
So the problem was in poor RT - actually there were no cDNA in positive control and all the signals were primer duplex. Re-performing RT helped.
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