New England Biolabs, M0681S, Induro® Reverse Transcriptase

Related Categories cDNA Synthesis & Reverse Transcriptases Applications RT-PCR,, cDNA Synthesis,, Reverse Transcription (cDNA Synthesis), Specification Materials Required but not Supplied Deoxynucleotide (dNTP) Solution Mix (NEB #N0447) RNase Inhibitor, Murine (NEB #M0314) Nuclease-free Water (NEB #B1500) FAQ Q: What is the difference between Induro Reverse Transcriptase (NEB #M0681) and ProtoScript II Reverse Transcriptase (NEB #M0368)? A: There are mainly two types of Reverse Transcriptases (RT): retrovirus-encoded RTs and intron-encoded RTs. Both RT types share some conserved domains and synthesize complementary DNA from an RNA template. The most common and well-characterized RTs are derived from retroviruses such as Moloney Murine Leukemia Virus (M-MuLV, MMLV), such as ProtoScript II Reverse Transcriptase. In contrast, Induro Reverse Transcriptase is a group II intron-encoded RT that exhibits higher thermostability, faster processivity, and better tolerance of reaction components when compared to retroviral RTs. Induro Reverse Transcriptase is useful for cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. Q: How do I choose between Induro Reverse Transcriptase and the different LunaScript and ProtoScript products? A: Induro Reverse Transcriptase is a group II intron-encoded RT that is useful for cDNA synthesis from long transcripts, RNAs with strong secondary structures, and RNA samples with inhibitors. In contrast, the LunaScript reagents are master mixes. The LunaScript RT Master Mix Kit (Primer-free) (NEB #E3025) is suitable for general cDNA synthesis. LunaScript RT SuperMix products (NEB #E3010/M3010) are optimized for two step RT-qPCR workflows where only short cDNA products are required. LunaScript RT SuperMix is also featured in the ARTIC SARS-CoV-2 sequencing workflow. This table outlines the applications and features of each. Intron-encoded RT Retroviral RT Products Induro™ Reverse Transcriptase (NEB #M0681) LunaScript® RT SuperMix Kit (NEB #E3010) LunaScript RT SuperMix (NEB #M3010) LunaScript RT Master Mix Kit (NEB #E3025) ProtoScript® II First Strand cDNA Synthesis Kit (NEB #E6560) Applications Long cDNA synthesis Impure RNA samples RNA-seq applications (e.g., direct RNA sequencing, long-read cDNA sequencing, tRNA seq Applications 2-step RT-qPCR detection] RNA-seq workflow Applications RNA-seq workflow 2-step RT-qPCR detection General cDNA synthesis 2-step RT-PCR detection Applications General cDNA synthesis RNA-seq workflow 2-step RT-PCR detection ✓ Short protocol ✓ Flexible choice of primers, modified dNTPs ✓ Higher reaction temperature ✓ Supermix ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Short protocol ✓ Tracking dye ✓ Higher reaction temperature ✓ Flexible choice of primers ✓ Kit format includes all necessary components Q: Can Induro Reverse Transcriptase be used in Oxford Nanopore Technologies® direct RNA sequencing? A: Yes, Induro Reverse Transcriptase is recommended by Oxford Nanopore Technologies for Direct RNA sequencing (SQK-RNA004) protocols. These protocols can be downloaded from the ONT Community pages (registration required). Q: What is the optimal reaction temperature for Induro Reverse Transcriptase? A: The optimal reaction temperature for Induro Reverse Transcriptase is 55°C. For difficult templates, the cDNA synthesis temperature can be increased up to 60°C. Q: Is it possible to inactivate Induro Reverse Transcriptase? A: Induro Reverse Transcriptase is thermostable but can be quickly inactivated by incubating at 95°C for 1 min. Alternatively, if downstream applications require maintaining the RNA integrity (e.g., direct RNA sequencing), an inactivation step of 70°C for 10 min can be used instead. In addition, Induro can be inactivated by protease digestion. For example, 1 µl of Thermolabile Protease K (NEB #P8111) can be added to the cDNA reaction. After an incubation at 37°C for 15 min, the Thermolabile Protease K can be inactivated by incubating at 55°C for 10 min. Q: What is the recommended incubation time for Induro Reverse Transcriptase? A: The recommended incubation time for Induro Reverse Transcriptase is 10 minutes at 55°C. Longer incubation times up to 30 minutes at 55°C may improve the yield of long cDNA products. Q: What is the longest cDNA generated by Induro Reverse Transcriptase? A: The longest cDNA generated is more than 20 kb based on direct cDNA sequencing data. Q: How much Induro Reverse Transcriptase should be used in a 20 µl reaction? Can the yield be further improved by using more enzyme? A: In general, 1 µl of Induro Reverse Transcriptase (200 units) is sufficient in a 20 µl reaction. The yield of some cDNA products may be increased by doubling the enzyme amount (400 units). Q: What RNA samples are compatible with Induro Reverse Transcriptase? A: For best results, we recommend an extraction or purification of the RNA starting material. However, Induro Reverse Transcriptase maintains its robust performance in the presence of many inhibitors. We recommend performing a pilot experiment for the specific sample/assay. Increasing the final MgCl2 (NEB #B9021) concentration by up to 3 mM at 1X may improve the inhibitor tolerance. Q: How much RNA template should I use? A: RNA templates can be total RNA, mRNA, or RNA synthesized by in vitro transcription. As a general rule, up to 1 μg of RNA template can be used in a 20 µl reaction. Q: What primer should I use with Induro Reverse Transcriptase? Can I spike in gene-specific primers to the cDNA reaction? A: For cDNA synthesis from RNAs with a poly(A) tail, the Oligo d(T)23 VN (NEB #S1327) is recommended. If desired, a longer dT oligo can be used. Alternatively, a gene-specific primer can be used in the cDNA reaction in place of the poly(dT) oligo. For RNA templates without a poly(A) tail, random primers or gene-specific primers can be used for cDNA synthesis. Addition of a gene-specific primer to the cDNA reaction may slightly increase the cDNA yield of the specific target. We have tested final concentrations of 0.1 µM, 0.5 µM, and 1 µM gene-specific primers with good performance at all concentrations, but would suggest 0.5 µM as a starting point and adjust accordingly. Q: What applications are best for Induro Reverse Transcriptase? A: Induro Reverse Transcriptase exhibits strong thermostability, processivity, and inhibitor tolerance. Induro may outperform other RTs in the following areas: Challenging cDNA synthesis from RNAs with strong secondary structures, long transcripts or impure samples Direct RNA-seq workflows on the Oxford Nanopore Technologies® platform Long-read cDNA sequencing Applications where intron-encoded RTs are used Q: Can the cDNA products generated by Induro Reverse Transcriptase be used in real-time PCR analysis? A: LunaScript RT SuperMix (NEB #E3010/M3010) has been optimized for 2-step RT-qPCR workflows and is recommended over Induro Reverse Transcriptase for this particular application. Q: What thermostable DNA polymerase can be used for PCR after cDNA synthesis? A: For downstream PCR, we recommend OneTaq 2X Master Mix (NEB #M0482 or M0485) for PCR detection up to 5kb, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) for highest fidelity, or LongAmp Taq 2X Master Mix (NEB #M0287) for high yields from longer products. Q: Why do I have low cDNA yields? A: There are several causes for low cDNA yield. Here are some suggestions to improve the yield: Check the integrity of the RNA by denaturing agarose gel electrophoresis or BioAnalyzer (Agilent). Intact RNA of high purity is essential for full-length cDNA synthesis. RNA should have a minimum A260/A280 ratio of 1.7 or a RIN number greater than 8. Ethanol precipitation followed by a 70% ethanol wash can remove contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides. An RNA purification step using RNA extraction kits or phenol/chloroform extraction can remove contaminant proteins such as proteases. Increase the amount of RNA used, especially for low abundant RNA. Q: Should I include a No-RT control reaction using Induro® RT for cDNA synthesis? A: Yes. The existence of residual amount of genomic DNA or carry-over products can interfere with downstream detection. Therefore, it is important to carry out the appropriate No-RT control reactions to account for these effects. Treating your RNA samples with DNaseI (NEB #M0303) will help remove contaminant DNA prior to performing cDNA synthesis. Q: How do I know whether my template RNA is of good quality? A: Intact RNA of high purity is essential for full-length cDNA synthesis. An absorbance ratio at A260/A280 above 1.7 or RIN number greater than 8 on a BioAnalyzer usually indicates RNA of high quality. Q: Can I store Induro Reverse Transcriptase and its reaction buffer at 4°C? A: For short-term storage, the enzyme can be kept at 4°C for one week and the 5X Induro Reaction Buffer can be stored at 4°C for up to one month. For long-term storage, it is recommended to store the enzyme and reaction buffer at -20°C. Q: Can I set up my cDNA synthesis reaction at room temperature when using Induro Reverse Transcriptase? A: Yes. Induro cDNA synthesis reactions can be set up at room temperature.