Hi guys,
i am going to test the expression level of a specific miRNA in some cell lines using Real time PCR. I am also intending to test the expression levels of some potential downstream targets using Real Time PCR also.
Since i am new to ordering issues,
i am wondering if i can use one syber green master mix for both detecting my miRNA and the downstream genes ?
I am specifically thinking of buying miRCURY LNA assays from qiagen and buying the syber greenmaster mix from applied biosystems ? Would they will be compatible together ? or shall i use the syber green master mix provided by qiagen in thei " miRCURY syber green PCR kit" ?
I looked for the two PCR protocols from applied biosystems and qiagen and i found that they are not even the same final reaction. However, I am still hoping i could buy the master mix from Applied biosystems so that it could be work for both the miRNA and the downstream target genes (if possible)
Could you please advice me ?
Hello Alaa,
Do you mean of "miRCURY LNA " is the cDNA synthesis kit for miRNAs? If yes, read below.
I've worked with "miRCURY LNA" and the quality is really satisfying.
I strongly recommend using "miRCURY SYBR green PCR kit". Once I tried to perform real-time PCR with SYBR Green from another company to evaluate the expression of my miRNA of interest and the results were disastrous while "miRCURY SYBR green PCR kit" showed perfect amplification in every sample.
To my experience, at least, use Qiagen SYBR miRCURY SYBR for miRNA amplification.
For the evaluation of downstream genes expression, I think you would be able to use Applied Biosystems SYBR. Of note. I'm not sure if miRNA cDNA synthesis kit works for mRNAs.
I don't think it makes a difference, but I would buy all reagents from the same vendor.
Hi Alaa,
The mircury system works quite well... retrotranscription is optimised for miRNAs to extend the length of the mature miRNA molecules, therefore you should use the primer assays by Exiqon as well. instead, you should be able to use any sybr green master mix, also from other companies. You should separately prepare another cDNA for analysing you mRNA targets of interest.
Good luck!
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