Efficient Reverse Transcription via Taqman Reagents leads to precise quantification of retroviral titer

June 17, 2015

University of Michigan
Pharmaceutical Sciences
Research Fellow

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Company:

Life Technologies

Product Name:

TaqMan® Reverse Transcription Reagents

Catalog Number:

N8080234

Quantification of retroviral titer can be tricky considering that they contain RNA as the genetic construct rather than DNA. Conversion of RNA to cDNA followed by Real-Time PCR is a very common strategy for viral titer quantification. This kit allows for quick and easy quantification when followed by a Real-Time PCR.

Experimental Design and Results Summary

Application

Retroviral Quantification

Starting Material

Isolated Viral RNA from retroviral sample for producer cell line - See http://www.clontech.com/US/Products/Viral_Transduction/Retroviral_Packaging/High-Titer_Retrovirus & https://www.qiagen.com/us/products/catalog/sample-technologies/rna-sample-technologies/viral-rna/qiaamp-viral-rna-mini-kit/

Protocol Overview

Simple protocol from the manufacturer was followed. However, optimization of reverse primer sequence and temperature and time of protocol is needed.

Tips

Make sure the lab bench is extremely clean. Use a cleaning agent to wipe down the table and micropipettes to get rid of genetic RNA floating around. Use filtered tips.

Results Summary

Good yield of cDNA were obtained which when further processed using a Real-Time PCR protocol were efficiently amplified with close to 100% efficiency. See attached figure of real-time amplification of 3 different dilution series of cDNA - (top left) - 2-fold dilution, (top right) - 5-fold dilution and (bottom left) - 10-fold dilution.

Additional Notes

None

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Summary

The Good

Quick Reverse Transciption.

The Bad

Reverse Transcriptase is expensive and is the limiting reagent. Optimization of reverse primers and reverse transcription conditions can be tricky.

The Bottom Line

Efficient cDNA conversion with very good amplification efficiency in Real-time PCR

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