RT-PCR Using Invitrogen's SuperScript III (SSIII) Synthesis System

February 01, 2017

Albert Einstein College of Medicine
Clinical Research
Pre-doctoral researcher

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Company:

Invitrogen by Thermo Fisher Scientific

Product Name:

SuperScript III First-Strand Sythesis System for RT-PCR

Catalog Number:

18080-051

We employed Invitrogen's SSIII RT-PCR kit to generate cDNA from mRNA that was previously isolated from whole blood using traditional phenol-chloroform extraction methods. The cDNA generated using the SSIII kit was used alongside SYBR green master mix in qRT-PCR gene expression studies. The SSIII kit is reliable and easy to use, and the included reagents allow for multiple annealing options (GSP, Oligo(dT), random hexamers) depending on the user's needs.

Experimental Design and Results Summary

Application

Reverse transcription

Starting Material

mRNA

Protocol Overview

First, incubate each mRNA sample with the appropriate primers and the dNTPs (included in each kit). Following the first incubation, combine the mRNA sample with a synthesis mix (buffer, salts, enzymes) and perform a second incubation for cDNA synthesis. After the second incubation is complete, remove any remaining mRNA with an RNase enzyme.

Tips

Use the incubation periods to prepare for subsequent steps to reduce experiment time. Leave enzymes at -20C until necessary and place on ice upon removal from frozen temperature.

Results Summary

cDNA concentrations can be determined using a NanoDrop. We measure the quality of our results based on our subsequent qRT-PCR data which includes melt curve evaluation.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

High quality cDNA product. Protocol is low stress, time efficient, and reliable.

The Bad

Expensive. Enzymes (the required reagents) run out before the remaining reagents included in the kit.

The Bottom Line

I would recommend this product for reverse transcription to anyone performing qRT-PCR. The manufacturer provides a detailed outline of the protocol and all required reagents are included in the kit.

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