Thermo Scientific Maxima Reverse Transcriptases (RTs) were developed through molecular evolution for optimal performance in cDNA synthesis. Our proprietary technology enabled the introduction of multiple favorable mutations that dramatically improved these enzymes' thermostability, robustness, and processivity to boost performance in cDNA synthesis.Maxima RTs are available in various formats for RT-PCR and RT-qPCR including as stand-alone enzymes and optimized kits and master mixes for ultimate convenience. Maxima RT kits and master mixes are also available with an integrated gDNA removal step for efficient, simplified workflows.

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Efficient reverse transcription in 2-step RT-qPCR

Our new Thermo Scientific Maxima H Minus cDNA Synthesis Master Mix offers consistently efficient and reproducible reverse transcription in 2-step RT-qPCR. Designed to deliver maximum value to your cDNA synthesis, the Maxima H Minus RT, an engineered, RNase H enzyme, has been formulated into this convenient one-tube master mix with a simplified protocol that enables consistency and maximum control of your RT-qPCR.

Why use a master mix format?

  • Enhances convenience in reaction set-up
  • Helps prevent pipetting variations due to multiple steps, enhances RT-qPCR data accuracy
  • Reduces risk of cross-contamination between samples

Why use Maxima H Minus RT?

  • Engineered MMuLV (Moloney murine leukemia virus) RT developed through molecular evolution
  • Improved thermostability
  • Faster synthesis rates
  • 50x increase in processivity
  • No RNase H activity
  • Demonstrates higher sensitivity than competitor products

High transcription efficiency 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 1). The good linearity strongly suggests reliable representation of relative quantities of different transcripts when using large or small amounts of input RNA.


Figure 1. 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 2). The higher transcription efficiency allows the use of less RNA and accurate detection of less expressed transcripts.

Figure 2. 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.

Consistent and reliable transcription across an array of targets

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 3).

Figure 3. 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.

Product features:

  • Enhances consistency of RT-qPCR results with increased efficiency of cDNA synthesis across a range of input RNA amounts
  • Controls pipetting variation with convenient master mix format
  • Controls gDNA in the RNA prep with included dsDNase and no-RT control for RT-qPCR

 

Technical details:

  • Consistently higher efficiency in cDNA synthesis across an array of targets in a 96-gene panel
  • High linearity in cDNA synthesis over a broad dynamic range (1 pg–1 µg of total RNA)
  • Maintains complete enzyme activity in reverse transcription from 42°C to 55°C

 

A complete cDNA synthesis workflow solution

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.

A Maxima No-RT control mix is also provided along with the Maxima H Minus RT Master mix to deliver a complete cDNA synthesis solution for two-step RT-qPCR. This No-RT control mix includes all the components of the reverse transcription master mix except the Maxima H Minus RT allowing the researcher to accurately confirm that there is no residual gDNA contamination.

Molecular evolution of Maxima and Maxima H Minus RTs

Thermo Scientific Maxima and Maxima H Minus RTs were developed through in vitro molecular evolution by introducing multiple mutations into traditional M-MuLV RT and selecting those that conferred favorable attributes for enhanced cDNA synthesis performance, including:

  • Superior yields of full-length cDNA
  • High reaction temperatures for improved transcription
  • High transcription efficiency on long RNA templates
     

Full-length cDNA over a wide temperature range

Maxima Reverse Transcriptases outperform other reverse transcriptases over a wide temperature range. Their tolerance of high reaction temperatures allows efficient transcription of RNA regions with extensive secondary structure and helps to improve primer specificity, resulting in high yields of full-length DNA (Figure 4).

 

Figure 4. High yields of cDNA over a broad temperature range. cDNA synthesis incorporating a radioactive label, using 1 μg of Invitrogen Millennium RNA markers (poly(A)-tailed) with oligo(dT)18 primer, was performed with Maxima H Minus Reverse Transcriptase and compared to cDNA synthesis performed with reverse transcriptases from other vendors according to the manufacturers’ protocols. Reaction products were resolved on an alkaline agarose gel.

Enhanced performance in long RT-PCR

Designed with proprietary mutations for enhanced performance, Maxima H Minus Reverse Transcriptase is capable of synthesizing full-length cDNA from very long RNA templates (Figure 5).

 

Figure 5. Amplification of long targets in two-step RT-PCR. Total RNA (1 μg) from mammalian cells was used in replicate reverse transcription reactions with Maxima H Minus Reverse Transcriptase and reverse transcriptases from other vendors, according to manufacturers’ recommendations. The resulting cDNA products were used as templates for PCR, and the results were visualized on agarose gels. Only Maxima H Minus Reverse Transcriptase was able to generate very long (13.3 kb) products with high yields.

Ideal for RT-qPCR─sensitive and reproducible quantification

Maxima Reverse Transcriptase is capable of reproducible cDNA synthesis from a wide range of template amounts, making it an ideal choice for RT-qPCR experiments (Figure 6). The premixed solutions in the Thermo Scientific Maxima First Strand cDNA Synthesis Kits help further improve reproducibility and save time during reaction setup.

 

Figure 6. Reproducible cDNA synthesis and low variability (<1% SD/Ct) with a wide range of starting RNA amounts. First-strand cDNA was synthesized from 100, 10, 1, 0.1, 0.01, and 0.001 ng of total RNA from mammalian cells using the Maxima First Strand cDNA Synthesis Kit in 16 replicate reactions each. Synthesized cDNA was used as template in qPCR with Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix. The amplification plot illustrates good consistency across the range of starting material.

Integrated gDNA removal step for simplified workflows

Maxima First Strand cDNA Kits include double-strand–specific DNase (dsDNase), which allows elimination of genomic DNA followed by cDNA synthesis, without any intermediate sample purification steps. The dsDNase is engineered to remove contaminating genomic DNA in 2 minutes without damaging RNA quantity or quality, or degrading single-stranded DNA such as primers and probes (Figure 7). The dsDNase is easily inactivated by a modest heat treatment (55°C). The subsequent genomic DNA–free RNA sample can be used directly in cDNA synthesis reactions, significantly simplifying the experimental workflow.

Figure 7. Effective removal of genomic DNA using dsDNase. Two-step RT-qPCR analysis of the PBGD gene with (RT+) and without (RT-) reverse transcriptase was plotted for several cDNA synthesis kits. cDNA synthesis was performed from 0.2 ng total Jurkat RNA using the Maxima First Strand cDNA Synthesis Kit with dsDNase, or kits from other vendors that offer gDNA removal. The orange, flat RT- plot for reactions using the Maxima kit indicates complete removal of contaminating gDNA, whereas RT- reactions using kits from other suppliers indicate amplification of residual gDNA.

How to perform gDNA removal and cDNA synthesis in a single tube

Efficient protection of RNA over a wide temperature range

RNase inhibitors are typically denatured at higher temperatures, releasing the RNases back into the reaction mix, which can then damage or degrade RNA. For protection of RNA against RNases, all Maxima First Strand cDNA Synthesis Kits include engineered, thermostable Thermo Scientific RiboLock RNase Inhibitor. The protein “locks” onto RNases and thus prevents RNA degradation at temperatures up to 55°C (Figure 8). The high thermostability of RiboLock RNase Inhibitor is important for successful reverse transcription at higher reaction temperatures.

Figure 8. RiboLock RNase Inhibitor efficiently protects RNA and inhibits up to 2 ng/20 μL of RNase A. Aliquots (20 μL) of total human RNA (1 μg) were supplemented at 37°C with 20 U of RiboLock RNase Inhibitor and increasing amounts of RNase A.
M: Thermo Scientific RiboRuler High Range RNA Ladder, ready-to-use
C: Total human RNA
1: Total human RNA with RNase A
2–5: Total human RNA with RiboLock RNase Inhibitor and RNase A

RiboLock RNase Inibitor exhibits high thermostability. Aliquots (20 μL) of total human RNA (1 μg) were supplemented with 20 U of RiboLock RNase Inhibitor and 50 pg of RNase A and incubated at increasing temperatures.

 

Understanding molecular evolution

The idea of using rational design criteria to improve enzyme performance is limited by our knowledge of polymerase fine structure and function. This limitation can be overcome by mimicking nature and using directed evolution to improve enzyme properties.

Our proprietary technique, compartmentalized ribosome display (CRD), allows for fast and efficient in vitro evolution of RTs [1, 2]. The technique has enabled the introduction and selection of multiple favorable mutations in wild type MMuLV RT, resulting in new, highly thermostable and processive RTs that supersede their wild type counterparts.

Molecular evolution by the CRD technique comprises several steps (Figure 9). First, beginning with the wild type MMuLV RT gene (1), an mRNA library is created based on random mutagenesis (2). Next, the mRNA library is translated in vitro to proteins that are associated with their mRNA progenitors (3). Then, the protein–mRNA complexes are placed into RT reaction mixtures and emulsified, yielding compartments containing one protein–mRNA complex each. Finally, the temperature is increased to create selective pressure under which only the improved mutants survive and produce full-length cDNA (4). By combining the best-performing mutations, highly processive MMuLV RT mutants capable of full-length cDNA synthesis at high temperatures were constructed.

  1. Baranauskas A et al. (2012) Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. PEDS. doi:10.1093/protein/gzs034
  2. Skirgaila R et al. (2013) Compartmentalization of destabilized enzyme–mRNA–ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes. PEDS. doi:10.1093/protein/gzt017
     

Figure 9. Key steps in the molecular evolution of MMuLV RT. 

Understanding RNase H activity

Wild type MMuLV RT possesses an RNA-dependent and DNA-dependent polymerase activity, and also an RNase H activity. The RNase H activity degrades RNA from RNA–DNA duplexes to allow efficient synthesis of dsDNA. However, with long mRNA templates, the RNA may be degraded prematurely, resulting in truncated cDNA. Hence, it is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning (Figure 10).

In contrast, RTs with intrinsic RNase H activity are often favored in qPCR applications, because they enhance the melting of RNA–DNA duplexes during the first cycles of PCR.

Figure 10. Comparison of RTs and cDNA synthesis with or without functional RNase H activity.

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  • How to perform RT and DNAse step in one tube with Maxima