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.
- Product finder
- Reaction size guidelines
- Real-time PCR products brochure
- Real-time PCR tutorial videos
- Guest webinar: extraction-free SARS-CoV-2 detection
- Guest webinar: developing and validating molecular diagnostic tests
- Unbiased preamplification of limited samples
- Accurate gene expression analysis with TB Green Premix Ex Taq II
- Efficient quantification of human gene expression with PrimeScript Reverse Transcriptase
- Rapid qPCR analysis from blood samples using PrimeScript Reverse Transcriptase
- Monitoring siRNA knockdown
- qPCR without optimization using TB Green Premix Ex Taq
- Fast synthesis of cDNA templates for real-time RT-PCR
- Specific, consistent real-time PCR with TB Green Premix Ex Taq II
- Mir-X microRNA quantification
An efficient method for quantification of human gene expression levels using PrimeScript reverse transcriptase
Data kindly provided by: Hiro Katagiri, Staff Scientist, NIH
Human GAPDH transcripts were more efficiently detected from cDNA generated with PrimeScript RT Master Mix than with the Company I kit.
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.
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.
Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2022 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.