Decorative reverse transcription

Thermo Scientific Maxima™ Reverse Transcriptase products are designed for consistency and efficiency in cDNA synthesis for RT-PCR and RT-qPCR applications.

  • Maxima Reverse Transcriptases and Maxima H Minus Reverse Transcriptases are available in a variety of formats including stand-alone enzymes, kits, and master mixes.
  • Maxima H Minus Reverse Transcriptase has diminished RNase H activity but retains all features of Maxima Reverse Transcriptase such as thermostability and enhanced processivity that have been improved. With these features, Maxima H Minus Reverse Transcriptase offers a robust performance in first strand cDNA synthesis.
  • Maxima H Minus Reverse Transcriptase and Maxima Reverse Transcriptase kits and master mixes are also available with an integrated gDNA removal step for efficient and simplified workflow.

Maxima H Minus Reverse Transcriptase product offerings

Maxima Reverse Transcriptase product offerings

Advantages of Maxima H Minus Reverse Transcriptase

Maxima H Minus Reverse Transcriptase is engineered with molecular evolution techniques selecting for mutations that confer dramatically improved thermostability, processivity, and activity rates compared to wild-type M-MuLV enzymes. These features support higher cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures. In RT-qPCR applications, Maxima H Minus Reverse Transcriptase also enables high efficiency synthesis over a wide range of input template amounts providing sensitive and accurate quantification of cDNA.
 

Thermostability

Maxima H Minus Reverse Transcriptase: Enhanced thermostability

Maxima H Minus Reverse Transcriptase outperforms other reverse transcriptases in terms of producing high yields of full-length cDNA over a wide temperature range (Figure 1). Its tolerance to high reaction temperatures allows efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity, thereby increasing overall yields.
Agarose gel electrophoresis results showing Maxima H Minus Reverse Transcriptase produced higher yields of cDNA over a wide temperature range when compared to competitor products
Figure 1. High yields of cDNA over a broad temperature range. cDNA synthesis was performed using (A) Maxima H Minus Reverse Transcriptase (20U) (B) Takara PrimeScript™ Reverse Transcriptase, (C) Promega GoScript™ Reverse Transcriptase, and (D) NEB ProtoScript™ II Reverse Transcriptase, according to each manufacturer’s protocol, and the experiments were performed at temperatures 42°C; 50°C; 55°C; 60°C; 65°C. 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primers were used. Reaction products were resolved by alkaline gel electrophoresis.
Processivity

Maxima H Minus Reverse Transcriptase: Increased processivity

Maxima H Minus RT has 50X increased processivity compared with wild type MMuLV reverse transcriptase enzymes. This reverse transcriptase is capable of synthesizing full-length cDNA from a wide range of RNA templates (Figure 2).

Gel showing the various size targets amplified by two-step RT PCR
Figure 2. Amplification of targets up to 20 kb in two-step RT-PCR. Total RNA (1 μg) from human cells (lanes 1 and 2) or mouse cells (lanes 3 and 4) were used in a reverse transcription reaction with Maxima H Minus Reverse Transcriptase according to manufacturer’s protocol. The resulting cDNA products were used as a template for PCR. Lane M: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.
Efficient cDNA synthesis

Maxima H Minus Reverse Transcriptase: Efficient cDNA synthesis

Maxima H Minus Reverse Transcriptase efficiently synthesizes cDNA from a wide range of template amounts, has higher yields, and better linearity in cDNA synthesis outperforming other commercially available reverse transcriptases, making it an exceptional choice for RT-qPCR experiments (Figure 3). The premixed solutions in the Thermo Scientific Maxima First Strand cDNA Synthesis Kits help further improve reproducibility and reaction setup time.

Figure 3. Consistently efficient reverse transcription over a wide range of input RNA amounts. Maxima H Minus First Strand cDNA Synthesis kit demonstrates consistently better reverse transcription efficiency than competitor kits. Amplification plots show variation of log (ΔRn) with PCR cycle number. RT-qPCR of human β-2 macroglobulin gene was performed from 10-fold serial dilutions of HeLa total RNA (1 μg to 1 pg). First strand cDNA was generated using the Maxima H Minus First Strand cDNA Synthesis kit and 7 other commercial First Strand cDNA Synthesis kits. cDNA was amplified using TaqMan Universal Master Mix II, with UNG on the Applied Biosystems ViiA7 Real-Time PCR System.

Linearity over a broad dynamic range

Maxima H Minus Reverse Transcriptase: Linearity over a broad dynamic range

The Maxima H Minus cDNA Synthesis Master Mix maintains high transcription efficiency and good linearity across a broad range of template concentrations (Figure 4). The observed linearity strongly suggests reliable representation of relative quantities of different transcripts when using large or small amounts of input RNA.

Figure 4. Broad dynamic range of Maxima H Minus cDNA Synthesis Master Mix. The standard curve illustrates high linearity (R2 = 0.999) across a broad range of input RNA, suggesting that the relative representation of specific RNA transcripts is preserved in the cDNA pool regardless of the abundance of total RNA. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix. cDNA was amplified using the Thermo Scientific Luminaris Probe qPCR Master Mix, low ROX, on the Applied Biosystems ViiA 7 Real-Time PCR System.
Consistent transcription efficiency

Maxima H Minus RT: Consistent transcription efficiency

The Maxima H Minus cDNA Synthesis Master Mix offers higher efficiency than reverse transcriptases from other suppliers at low and high input RNA amounts (Figure 5). The higher transcription efficiency allows the use of less RNA and accurate detection of less expressed transcripts.

Amplification plot demonstrates efficient transcription by Maxima H Minus cDNA Synthesis Master Mix when compared to competitor products
Figure 5. Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix. The Maxima H Minus cDNA Synthesis Master Mix demonstrates better efficiency over a wide range of input RNA amounts when compared to competitor’s RTs. Amplification of the human 18S RNA gene was performed on 10-fold serial dilutions of HeLa total RNA (1 μg to 0.1 pg). First-strand cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix, Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR, and reverse transcriptases from four other suppliers. cDNA was amplified using the Luminaris Probe qPCR Master Mix, low ROX on the ViiA 7 Real-Time PCR System. Amplification plots indicate variation of ΔRn with cycle number.
Reliable transcription

Maxima H Minus Reverse Transcriptase: Reliable transcription across an array of targets

The Maxima H Minus cDNA Synthesis Master Mix shows consistently better efficiency than reverse transcriptase master mixes from other suppliers over a range of 96 target genes in RT-qPCR when normalized to data generated with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Figure 6).

Figure 6. Consistently lower Ct values. Maxima H Minus cDNA Synthesis Master Mix shows higher cDNA synthesis efficiency compared to other commercial master mixes over a wide range of target genes. Maxima H Minus cDNA Synthesis Master Mix was compared to Thermo Scientific Maxima First Strand cDNA Synthesis Kit for RT-qPCR and master mixes from other suppliers using 96-gene Applied Biosystems TaqMan Assay panels with 100 ng HeLa total RNA input. Using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR as the reference, the ΔCt values (ΔCt = Ct Maxima H Minus cDNA Synthesis Master Mix or other commercial product – Ct Maxima First Strand cDNA Synthesis Kit for RT-qPCR) are shown for each of the 96 genes in the panel.
Integrated gDNA removal

Easy, integrated gDNA removal step

The double-strand-specific DNase (dsDNase) enables faster, efficient, and complete gDNA removal compared to conventional DNase I (Figure 7). dsDNase-treated RNA samples show no decrease in RNA integrity or quantity. With the use of dsDNase treatment prior to cDNA synthesis, the risk of sample loss can be minimized.

Figure 7. Integrated dsDNase enables removal of gDNA in 2 minutes.

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