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Home › Learning centers › Real-time PCR › Technical notes › Efficient quantification of human gene expression with PrimeScript Reverse Transcriptase

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Tech Note

An efficient method for quantification of human gene expression levels using PrimeScript reverse transcriptase

Data kindly provided by: Hiro Katagiri, Staff Scientist, NIH

Introduction Results Conclusions Methods

Introduction  

PrimeScript RT Master Mix (Perfect Real Time) is a high-efficiency reverse transcriptase mix that includes all of the reagents required for cDNA synthesis. This experiment compared the performance of PrimeScript RT Master Mix to a cDNA synthesis kit from Company I in measuring expression of human GAPDH by quantitative PCR (qPCR). GAPDH was detected at higher levels from cDNA generated with PrimeScript RT Master Mix than from cDNA generated with the alternative kit, illustrating the superior reverse transcription efficiency of PrimeScript RT Master Mix.

Results  

Human GAPDH transcripts were more efficiently detected from cDNA generated with PrimeScript RT Master Mix than with the Company I kit.

Expression of GAPDH as determined by qPCR. Expression levels of human GAPDH generated with kits from Company I or PrimeScript RT Master Mix. Heat denatured samples (blue bars) show similar expression levels to samples what were not heat denatured (orange bars). Both PrimeScript RT Master Mix and the Company I reverse transcriptase kit generated cDNA regardless of heat denaturation of the RNA template.

Comparing Terra PCR Direct to other PCR enzymes for amplification from crude samples.

PCR amplification of GAPDH. PCR amplified products generated with primers specific for human GAPDH are found in all samples. Lanes 1 and 2 represent amplification from cDNA generated with Company I's kit, and lanes 3 and 4 represent amplification from cDNA generated with PrimeScript RT Master Mix. Lanes 1 and 3 were heat denatured samples, while lanes 2 and 4 were not heat denatured.

Conclusions  

PrimeScript RT Master Mix was able to quickly (in ~15 minutes) and efficiently generate cDNA, which could be used to quantify the expression of human GAPDH, a 'housekeeping' gene commonly used to normalize expression levels. Higher levels of GAPDH were detected from cDNA samples generated with PrimeScript RT Master Mix kit than with Company I's kit, showing the greater sensitivity of detection that can be achieved, in less time, with PrimeScript RT Master Mix.

Methods  

Total RNA was isolated from HEK 293 cells using an Absolutely RNA Miniprep Kit (Agilent) according to the recommended protocol. Approximately 1 µg of RNA was used as a template for reverse transcription.

PrimeScript RT Master Mix was compared to a cDNA synthesis system for reverse transcription PCR from Company I. RNA treatment conditions (with and without heat denaturation at 65°C for 5 min) were also tested. For Company I, heat denaturation was performed in the presence of RNA, primers, dNTPs, and DEPC-treated water. For PrimeScript RT Master Mix, RNA was heat-denatured in DEPC-treated water. Reverse transcription was performed according to the recommended protocols.

One microliter of the reverse transcriptase reaction was used directly for PCR or qPCR using primers specific for human GAPDH. PCR was performed using TaKaRa Ex Taq polymerase according to the recommended protocol. qPCR was performed with iQ SYBR Green Supermix (Bio-Rad). Two standard curves were prepared using a plasmid containing human GAPDH cDNA; serial dilutions of the plasmid were prepared using either DEPC-treated water or EASY Dilution (for Real Time PCR). Products of the reverse transcriptase reactions were analyzed in triplicate.

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Cat. # Product Size Price License Quantity Details
RR036A PrimeScript™ RT Master Mix (Perfect Real Time) 200 Rxns USD $477.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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RR036A: PrimeScript RT Master Mix (Perfect Real Time)

RR036A: PrimeScript RT Master Mix (Perfect Real Time)
RR036B PrimeScript™ RT Master Mix (Perfect Real Time) 800 Rxns USD $1531.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays. Cat. # RR036B contains 4 of Cat. # RR036A. Please refer to Cat. # RR036A for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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