Maxima Reverse Transcriptases

Thermo Scientific Maxima Reverse Transcriptases (RTs) offer robust performance in cDNA synthesis for optimal value. Maxima RT is designed for enhanced consistency and efficiency in cDNA synthesis for both RT-PCR and RT-qPCR applications in a variety of formats (e.g., stand-alone enzymes, RT kits and master mixes).  Maxima RT kits and master mixes are also available with an integrated gDNA removal step for efficient and simplified workflow.

Developed through molecular evolution, our latest Maxima H Minus reverse transcriptase is an enzyme with diminished RNase H activity but contains all the features of Maxima RT with high thermostability and enhanced processivity.  With these improved features, Maxima H Minus reverse transcriptase offers a robust performance in first strand cDNA synthesis.

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Feature video: Maxima first strand cDNA synthesis kits in action

Thermo Scientific Maxima First Strand cDNA Synthesis Kits with double-stranded DNase combine genomic DNA elimination and cDNA synthesis into a simple, one tube workflow that can be completed in as few as 15 minutes.

Maxima H Minus RTs are engineered with molecular evolution techniques selecting for mutations that confer dramatically improved thermostability, processivity and robust activity rates compared with wild-type M-MuLV enzymes. These features support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures.

Maxima H Minus RT also shows greater efficiency with synthesis of full length cDNA templates. It lacks RNase H activity that normally degrades RNA from RNA-DNA duplexes as the cDNA is being synthesized; which helps to prevent premature degradation of RNA. In RT-qPCR applications, Maxima H Minus RT also enables high efficiency synthesis over a wide range of input template amounts providing sensitive and accurate quantification of cDNA.

High thermostability

Maxima H Minus Reverse Transcriptase outperforms other reverse transcriptases, producing high yields of full-length cDNA over a wide temperature range (Figure 1). Its tolerance of high reaction temperatures allows for efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity increasing overall yields.

High yields of cDNA over a broad temperature range
Figure 1. High yields of cDNA over a broad temperature range. cDNA synthesis was performed using 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primers and (A) Maxima H Minus RT (20U) following the recommended  protocol across a temperature range (42 °C; 50 °C; 55 °C; 60 °C; 65 °C).  Comparable reactions were performed with RTs from other suppliers including (B) Takara PrimeScript RT, (C) Promega GoScript RT, and (D) NEB ProtoScript II RT, according to each manufacturer’s protocol. Reaction products were resolved by alkaline gel electrophoresis.

High processivity

Designed with proprietary mutations for enhanced processivity, Maxima H Minus RT has 50x increase in processivity compared with wild type MMuLV RT enzymes. This RT is capable of synthesizing full-length cDNA from a wide range of RNA templates (Figure 2).

Amplification of targets up to 20 kb in 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 RT reaction with Maxima H Minus RT following the maufacturer’s recommended protocol. The resulting cDNA products were used as a template for PCR. M: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.

Efficient cDNA synthesis

Maxima H Minus RT is capable of efficient cDNA synthesis from a wide range of template amounts, outperforming other suppliers’ RTs with higher yields and better linearity in cDNA synthesis making it an ideal 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 save time during reaction setup.

Consistently efficient RT over a wide range of input RNA amounts
Figure 3. Consistently efficient RT over a wide range of input RNA amounts. Maxima H Minus First Strand cDNA Synthesis kit demonstrates consistently better reverse transcription efficiency than other suppliers’ 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.

Maxima H Minus RT has been formulated into a convenient one-tube master mix with a simplified protocol that enables consistency and maximum control of your RT-qPCR.

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

The Maxima H Minus cDNA Synthesis Master Mix offers higher efficiency than RTs 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.

Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix

Figure 5. Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix. The Maxima H Minus cDNA Synthesis Master Mix demonstrates better efficiency than other suppliers’ RTs over a wide range of input RNA amounts. 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 RTs 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 across an array of targets in a 96-gene panel

The Maxima H Minus cDNA Synthesis Master Mix shows consistently better efficiency than RT 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 targets. 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 – CtMaxima First Strand cDNA Synthesis Kit for RT-qPCR) are shown for each of the 96 genes in the panel.
 

Easy, integrated gDNA removal step

The Maxima H Minus cDNA Synthesis Master Mix is available with a double-strand–specific DNase (dsDNase), which enables faster, more efficient, and complete gDNA removal compared with conventional DNase I. dsDNase-treated RNA samples show no decrease in RNA integrity or quantity.  With the use of dsDNase treatment prior to cDNA synthesis, you can minimize the risk of sample loss observed following sample clean-up with conventional DNase I treatment.

Maxima H Minus RTs are engineered with molecular evolution techniques selecting for mutations that confer dramatically improved thermostability, processivity and robust activity rates compared with wild-type M-MuLV enzymes. These features support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures.

Maxima H Minus RT also shows greater efficiency with synthesis of full length cDNA templates. It lacks RNase H activity that normally degrades RNA from RNA-DNA duplexes as the cDNA is being synthesized; which helps to prevent premature degradation of RNA. In RT-qPCR applications, Maxima H Minus RT also enables high efficiency synthesis over a wide range of input template amounts providing sensitive and accurate quantification of cDNA.

High thermostability

Maxima H Minus Reverse Transcriptase outperforms other reverse transcriptases, producing high yields of full-length cDNA over a wide temperature range (Figure 1). Its tolerance of high reaction temperatures allows for efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity increasing overall yields.

High yields of cDNA over a broad temperature range
Figure 1. High yields of cDNA over a broad temperature range. cDNA synthesis was performed using 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primers and (A) Maxima H Minus RT (20U) following the recommended  protocol across a temperature range (42 °C; 50 °C; 55 °C; 60 °C; 65 °C).  Comparable reactions were performed with RTs from other suppliers including (B) Takara PrimeScript RT, (C) Promega GoScript RT, and (D) NEB ProtoScript II RT, according to each manufacturer’s protocol. Reaction products were resolved by alkaline gel electrophoresis.

High processivity

Designed with proprietary mutations for enhanced processivity, Maxima H Minus RT has 50x increase in processivity compared with wild type MMuLV RT enzymes. This RT is capable of synthesizing full-length cDNA from a wide range of RNA templates (Figure 2).

Amplification of targets up to 20 kb in 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 RT reaction with Maxima H Minus RT following the maufacturer’s recommended protocol. The resulting cDNA products were used as a template for PCR. M: Thermo Scientific GeneRuler 1 kb Plus DNA Ladder.

Efficient cDNA synthesis

Maxima H Minus RT is capable of efficient cDNA synthesis from a wide range of template amounts, outperforming other suppliers’ RTs with higher yields and better linearity in cDNA synthesis making it an ideal 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 save time during reaction setup.

Consistently efficient RT over a wide range of input RNA amounts
Figure 3. Consistently efficient RT over a wide range of input RNA amounts. Maxima H Minus First Strand cDNA Synthesis kit demonstrates consistently better reverse transcription efficiency than other suppliers’ 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.

Maxima H Minus RT has been formulated into a convenient one-tube master mix with a simplified protocol that enables consistency and maximum control of your RT-qPCR.

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

The Maxima H Minus cDNA Synthesis Master Mix offers higher efficiency than RTs 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.

Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix

Figure 5. Enhanced transcription efficiency of Maxima H Minus cDNA Synthesis Master Mix. The Maxima H Minus cDNA Synthesis Master Mix demonstrates better efficiency than other suppliers’ RTs over a wide range of input RNA amounts. 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 RTs 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 across an array of targets in a 96-gene panel

The Maxima H Minus cDNA Synthesis Master Mix shows consistently better efficiency than RT 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 targets. 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 – CtMaxima First Strand cDNA Synthesis Kit for RT-qPCR) are shown for each of the 96 genes in the panel.
 

Easy, integrated gDNA removal step

The Maxima H Minus cDNA Synthesis Master Mix is available with a double-strand–specific DNase (dsDNase), which enables faster, more efficient, and complete gDNA removal compared with conventional DNase I. dsDNase-treated RNA samples show no decrease in RNA integrity or quantity.  With the use of dsDNase treatment prior to cDNA synthesis, you can minimize the risk of sample loss observed following sample clean-up with conventional DNase I treatment.

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