Choosing the Right Reverse Transcriptase
We offer a range of quality reverse transcriptases for first-strand cDNA synthesis, end-point RT-PCR and RT-qPCR. Here we summarize the features of each reverse transcriptase to help you find the best enzyme for your needs.
- Reverse Transcriptase Comparison
- GoScript™ RT
- M-MLV RT, RNase H- Pt. Mut.
- AMV RT
Robust, Reliable cDNA Synthesis for qPCR
GoScript™ Reverse Transcriptase is a highly active, high-value enzyme optimized to give excellent performance in quantitative RT-PCR. Its robust performance also makes it ideal for most RT-PCR applications.
- Ultra Active: Saves money on every reaction
- Sensitive: Detects rare transcripts
- Processive: Transcribes long messages
- Resilient: Synthesizes cDNA in the presence of strong inhibitors
- Replaces SuperScript® III
Detect Rare or Abundant Transcripts
GoScript™ Reverse Transcriptase cDNA synthesis coupled with GoTaq® qPCR Master Mix can provide detection of input templates over a 9-log order dynamic range.
Transcribe Long Messages More Effectively
Reverse transcription of an 8.9kb APC transcript. Human colon total RNA (Clontech Laboratories, Inc.) was reverse transcribed using oligo(dT) (O) or random primers (R). cDNA was analyzed by endpoint PCR using GoTaq® Green Master Mix (Cat.# M7122) with primers for a 940bp amplicon at the 5´ end of the transcript. Each reaction (5µl) was analyzed by agarose gel electrophoresis and ethidium bromide staining. All samples shown were analyzed in the same data set and gel with the same scan settings. Gel images are representative of results from three separate experiments
Get Better Performance in the Presence of PCR Inhibitors
Sensitivity of reverse transcriptases to ethanol inhibition. An in vitro-transcribed RNA (1.2kb Kanamycin Positive Control RNA, Cat.# C1381) was reverse transcribed using oligo(dT) primer according to the manufacturer's instructions for each RT. Reverse transcription was carried out in the absence and presence 5%, 10% and 20% ethanol. cDNA was analyzed by real-time PCR using GoTaq® qPCR Master Mix (Cat.# A6001). The change in quantification cycle values in the absence and presence of ethanol was determined and converted to relative change in transcript level [2(Cqinhibitor–Cqnone)]. Data are the average of three separate experiments.
A High-Performance Reverse Transcriptase for Daily Use
Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]), Point Mutant, is an RNA-dependent DNA polymerase that can be used in cDNA synthesis as well as primer extension.
M-MLV RT RNase H– Point Mutant Highlights:
- RNase H–: Optimal conditions for preparing full-length cDNA from long RNA templates.
- Thermal Stability: Prevents problems associated with secondary structure.
- Increased Polymerase Activity: Higher cDNA yields and better results with low amounts of RNA template.
- Replaces SuperScript® II
Thermal Stability and Synthesis of Large Transcripts
cDNAs were synthesized from 1µg of total human RNA using M-MLV (H–), Point Mutant, (Panel A) or SuperScript® II (Panel B) reverse transcriptase at 37°C, 42°C and 50°C. GoTaq® Long PCR amplifications were performed on each transcription reaction using five primer sets, amplifying products from 9.4kb to 1.5kb. Ten microliters of the 9.4, 6.9, and 5.2kb PCR products and 1µl of the 3.2 and 1.5kb PCR products were separated on a 1% agarose gel. Lane M. BenchTop 1kb DNA Ladder (Cat.# G7541).
M-MLV RNase H– Generates High-Quality cDNA from Low Amounts of RNA Template
Total human RNA was serially diluted (100ng–100fg) and used as template in reverse transcription reactions with M-MLV (H–), Point Mutant,( Panel A) or SuperScript® II (Panel B) reverse transcriptase. GoTaq® PCR amplification was performed on the transcripts using GAPDH-specific primers (0.5kb product size). Twenty microliters of the PCR products were separated on a 1% agarose gel. Lane M. BenchTop 1kb DNA Ladder (Cat.# G7541).
M-MLV, RNase H– is Equivalent to SuperScript® II
Sensitivity in qPCR. Kanamycin control RNA was serially diluted, spiked into 1ng of total human RNA and used as template in reverse transcription reactions. Quantitative PCR using GoTaq® qPCR System with kanamycin control primers was performed using 5µl of the product. N=3.
A Great Choice for Difficult Templates With High Secondary Structure
- Thermal Stability: AMV RT is the preferred reverse transcriptase for templates with high secondary structure due to its stability at higher reaction temperatures (37–58°C).
AMV RT Reverse Transcriptase displays optimal activity at a higher temperature than M-MLV RT (48 versus 37°C, respectively), making it particularly useful for reverse transcription of RNA templates that exhibit significant secondary structure. However, AMV RT also has a stronger intrinsic RNase H activity, making generation of longer (>5kb) cDNAs more difficult.
Single-Cell RT-PCR using AMV RT
Representative 2% agarose gel showing PCR products following laser capture microdissection and single-cell RT-PCR. TH = tyrosine hydroxylase; βA = beta actin; (+) = positive control; (--) = negative control. The negative control represented in lane 4 lacked RNA; the negative control represented in lane 8 lacked AMV RT. Positive control (lanes 3 and 7) included total brain RNA.