I am currently doing miRNA research and got the miRCURY LNA RT kit from Qiagen for RT reaction to turn my RNA into cDNA.
A few papers have used SYBR green kit from another brand and not the Qiagen miRCURY LNA SYBR Green PCR kit, and that is why I got the kit, but they did not specify the dilution of the cDNA they used in the qPCR reaction.
Does anyone have experience with this? please kindly help.
Many Thanks!
I had a similar experience with Advanced miRNA cDNA Synthesis Kit (Applied Biosystems). I used these cDNA in the PCR reaction with qPCRmix-HS SYBR+HighROX (Eurogene). I used dillution of cDNA 1:25 (before additional step "amplification") and dillution 1:40 (after "amplification", which resulted in more copies of cDNA), the last one was recomended by the manufacturer with their PCR mix. These dillutions showed good results with SYBR-green PCR mix with primers designed by miRprimer software. However, next time I would use more concentrated cDNA that were synthesized without addaitional "amplification".
In addition, I adapted the time and temperature of the PCR steps, combining the recommendations for microRNA (shorter time) and for my PCR kit (optimal temperatures and enzyme working time).