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Thermo Scientific Maxima Reverse Transcriptase (RT) was developed through in vitro evolution of M-MuLV RT. The enzyme possesses an RNA-dependent and DNA-dependent polymerase activity as well as RNase H activity. The engineered enzyme features dramatically improved thermostability and robustness, and increased synthesis rates compared to wild type M-MuLV RT.
Maxima Reverse Transcriptase is capable of reproducible cDNA synthesis from a wide range of input total RNA amounts (from 1 pg to 5 μg) at elevated temperatures (50 to 65°C), which makes this enzyme an ideal tool for two step RT-qPCR (see supporting data).
Due to its high thermostability, Maxima enzyme maintains full activity during the entire reverse transcription reaction, generates the highest yields of cDNA, and is able to synthesize even very long RNA transcripts up to 20 kb (see supporting data). The reaction temperature can be increased up to 65°C for efficient transcription of RNA regions with a high secondary structure or to improve specificity using gene specific primers. Due to its increased synthesis rate, the reverse transcriptase reaction can be completed in 15 to 30 min.
Also available: Maxima First Strand cDNA Synthesis Kit for RT-qPCR
Figure 1 | High thermostability of Maxima Reverse Transcriptase at 50°C. Reverse transcriptases were incubated in 1X reaction buffer. At the indicated time points (5 to 240 minutes), enzyme activity was determined in a standard activity assay.
Figure 2 | Amplification of targets up to 20 kb in two step RT-PCR. 1 µg of total RNA from Jurkat cells (1 and 2) or total RNA from mouse (3 and 4) were used in a reverse transcription reaction with Maxima
Reverse transcriptase and other RTs in the optimal conditions for each enzyme. Synthesized cDNA was used as a template in PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:
1 – 6.8 kb POLE (human polymerase)2 – 9.4 kb FBN1 (human fibrillin 1)3 – 13.3 kb Dmd (mouse dystrophin)4 – 20.0 kb Neb (mouse nebulin)M – GeneRuler 1 kb Plus DNA Ladder (#SM1331)
Figure 3 | High yields of cDNA over a broad temperature range. Synthesis of 33P-labeled cDNA was performed using 200 units of each reverse transcriptase, 1 µg of Ambion RNA Millennium markers and oligo(dT)18 primer at various temperatures. Reaction products were analyzed on a 1% alkaline agarose gel.
Figure 4 | High cDNA synthesis rate at 50°C. Synthesis of cDNA was performed at 50°C for 5, 15, 30, and 60 minutes using 1 µg of 7 kb RNA transcript as a template. Reaction products were analyzed on a 1% alkaline agarose gel. Only Maxima RT was able to complete synthesis of 7 kb transcript in 5 minutes.
Figure 5 | Reproducible RT-qPCR results using Maxima Reverse Transcriptase. 100 ng to 1 pg of total Jurkat cell RNA and a mix of Oligo(dT)18 and random hexamer primers were used with Maxima RT in 16 replicate reactions.
Synthesized cDNA was used as a template in subsequent qPCR with Maxima SYBR Green/ROX qPCR Master Mix (#K0221) on the ABI 7500 Real-Time PCR instrument. Parallel reactions with Maxima RT demonstrate reproducible cDNA synthesis and low variability levels ( < 1% SD/Cq) with a wide range of starting RNA amounts.