An Efficient Method for Quantification of Human Gene Expression Levels Using PrimeScript Reverse Transcriptase
Data kindly provided by: Hiro Katagiri, Staff Scientist, NIH
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 for measuring the expression level 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.
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.
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.
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.