Hello, I have prepared miRNA cDNA from 50ng RNA and based on the kit (Qiagen miRCURY LNA miRNA Assay) it is suggested i perform 60X dilution of the cDNA whish would bring down my RNA conc. to nearly 0.8 ng/ul. Please suggest on the dilution of the cDNA.
Dear H Surachandra Singha
Dilution/amount/volume/concentration referred by Qiagen for both RNA and cDNA do not works as per instructions since your choice of miRNA conc. itself will be vary.
Since, the Kit efficiency high so you can use 300-1000ng of total RNA for miRNA cDNA conversion and go for qRT-PCR after diluting the cDNA into 1:10 or 1:20 ratio. It will work, practically the amount of miRNA is less in cells so to detect such low amount, the dilution referred by Qiagen do not works. You need to standardize the conc.
As as products, it wonderful and we are using it for last 2yr.
Cheers!!!
When I have amplified specific genes from miRNA, because the amount tends to be very low I generally dilute nby no more than 1:10
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