I am using miScript RT II Kit for cDNA preparation from total RNA pool, this kit provides two buffers hispec and hiflex. on QPCR lower ct values comes with hiflex but not with hiSpec. which one to use for mature miRNA quantification, the kit says hiSpec but the ct values it provide are way beyond 35?
What kind of starting materials do you have?
Do you check the quality of your RNA? (even if miRNAs are very stable it is always better to work with good quality material)
How much RNA do you put into your cDNA reaction?
And how do you dilute your cDNA afterwards?
I also use the same kit and I use the HiSpec buffer. Normally I put 500 ng RNA into the cDNA reaction and dilute the cDNA then about 1:50. This works quite well for me.
In your place I would run a cDNA dilution series with an ubiquitously expressed miRNA in a qPCR to identify the optimal dilution.
You can also mix your cDNA with plasmid DNA and then use it as a template to check if any inhibiting substances are present. If you would have more than 3 cycles difference from the plasmid only ct then dilute these samples and repeat qPCR.
Hope I could help you a litte bit.
Thank for the suggestion
strating material is 20 buffalo oocytes
from 20 oocytes i get approx 10-40 ng/ul . i fix my RNA at 50ng/cDNA reaction , but i do not dilute the cDNA for qPCR Reaction, by doing so it comes in the given range of my quantitect SYBR green reaction that is 50pg-3ng.
but why it comes great with the HiFlex Buffer??
I use HiFlex buffer for mature miRNA quantification
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