Universal cDNA Synthesis for microRNA studies

January 19, 2015

Institute of Cellular Medicine
Haematological Sciences
PhD Student

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Exiqon

Product Name:

Universal cDNA Synthesis Kit II

Catalog Number:

203301

The main aim of the research was to validate a set of microRNAs that were identified via a global profiling experiment in clinical skin biopsies and test that they retained their significance in differentiating between the different stages of the disease. Clinical skin biopsies are very precious and thus there are very limited samples for use. To maximize samples the Universal cDNA Synthesis Kit II was ideal for making cDNA and then utilizing it for downstream RT-qPCR applications. Moreover, the universal synthesis allowed all the microRNAs to be quantified in the same cDNA thereby reducing technical variations.

Experimental Design and Results Summary

Application

MicroRNA cDNA synthesis

Starting Material

Total RNA

Protocol Overview

All reagents and reaction steps for the cDNA step were performed on ice. First strand cDNA was synthesized by diluting total RNA samples (5 ng/µl) using nuclease-free water. The universal cDNA master mix (Product No: 203301) comprised of: 5X reaction buffer, nuclease-free water, enzyme, and the diluted total RNA. The thermal cycler (Applied Biosystems, 2720 Thermal Cycler) reactions set up were as follows: 42 degrees Celsius for 60 minutes, 95 degrees Celsius for 5 minutes and then samples stored at -4 degrees Celsius till use.

Tips

Store the cDNA as synthesized and only dilute it when needed for RT-qPCR reactions.

Results Summary

The cDNA synthesized was then used for downstream microRNA RT-qPCR using specific primers for the reactions. The cDNAs were specific and this was clearly evident from the single peaks observed in the dissociation curves.

Additional Notes

None

Related Categories

Image Gallery

Summary

The Good

Universal cDNA, specific, sensitive, and easy to use.

The Bad

None

The Bottom Line

It is one of the best microRNA cDNA synthesis kits that I have used till now and would highly recommend it to other researchers, in particular, to those in the biomarker discovery field.

Join the discussion