New England Biolabs, M0466S, Template Switching RT Enzyme Mix

The Template Switching RT Enzyme Mix and accompanying reaction buffer enable efficient template switching activity in a reverse transcription reaction. The mix contains a unique RT and Murine RNase Inhibitor. Unlike competitor RT products, no additives (such as PEG or betaine) are required for optimal performance, simplifying reaction setup. In conjunction with a template switching oligo (TSO), cDNA is synthesized with a known sequence of choice attached to the 3′ end. The resulting cDNA can be amplified by PCR or serve as template for 5′ RACE (rapid amplification of cDNA ends) or 2 Related Categories cDNA Synthesis & Reverse Transcriptases,, PCR, qPCR & Amplification Technologies Applications cDNA Synthesis,, Reverse Transcription (cDNA Synthesis) Specification Heat Inactivation 80°C FAQ Q: Do I need to add RNase Inhibitor to the RT reaction when using the Template Switching RT Enzyme Mix? A: The Template Switching RT Enzyme Mix contains Murine RNase Inhibitor. If the starting material is RNA, no additional RNase Inhibitor is required. If the starting material is cells/tissues, please follow the instructions for cell lysis or RNA isolation, which may require RNase inhibitor. Q: Numerous publications describe the addition of various additives (e.g., betaine and PEG) to improve template switching. Can I add any of those to my reaction? A: The Template Switching RT Enzyme Mix and Buffer have been extensively optimized for template switching. Because this development has already been done, we generally observe the best performance in the reagents as provided. Q: Is the Template Switching RT Enzyme Mix compatible with RT primers with degenerate bases for cDNA synthesis and amplification, as described in the mcSCRB method [1]? A: We have compared the performance of the Template Switching RT Enzyme Mix and Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). The cDNA libraries made with the NEB Template Switching RT Enzyme Mix contain at least 10% more transcripts than those made with Maxima H Minus supplemented with 7.5% PEG as described in the mcSCRB method. In addition, the NEB libraries exhibited improved RNA base distribution metrics as listed below: RT Options % Exon % rRNA % Intron % Intergenic NEB 77.8±0.3 2.1±0.3 10.3±0.0 9.7±0.0 mcSCRB 50.0±1.8 24.8±1.8 9.8±0.2 15.4±0.2 1 Bagnoli, JW. et al. (2018) Nat Commun. 2937-2944 . Q: What type of template switching oligos (TSOs) are compatible with the Template Switching RT Enzyme Mix? A: In general, different TSOs can be used including TSOs with 5′ modifications, such as biotin, isomeric bases or abasic spacers. We recommend a TSO with rGrGrG at the 3′ end for efficient template switching but various types/sequences of TSOs are compatible with the Template Switching RT Enzyme Mix. Note that for downstream amplification using archaeal family B-type polymerases (e.g. Q5® and Phusion®), a TSO containing rU residues may cause inhibition. For a specific application, please refer to General Guideline/TSO in the respective protocol. Q: Can I use the Template Switching RT Enzyme Mix for regular RT reactions? A: This product can also be used in a more general first strand synthesis reaction by omitting the TSO, but it is likely that other products would be better suited for these reactions. For recommended products by application, please visit: https://www.neb.com/tools-and-resources/selection-charts/cdna-synthesis-selection. Q: What is the efficiency of template switching when using the Template Switching RT Enzyme Mix? A: The template switching efficiency varies based on target sequences. We typically observe an efficiency between 20-60% as measured by qPCR. Q: Does the Template Switching RT Enzyme Mix work for prokaryotic RNA? A: Yes, the Template Switching RT Enzyme Mix can be applied to prokaryotic RNA. However, the template switching efficiency for prokaryotic RNA, whose primary transcripts contain 5’-PPP, will be lower compared to eukaryotic mRNA with a cap structure. Q: Does the Template Switching RT Enzyme Mix work for fragmented RNA? A: The suitability of fragmented RNA as a starting material will depend on the application/question of interest. For 5′ RACE and 2nd strand cDNA synthesis, intact RNA inputs are required to retain the full transcription start site. For cDNA amplification, fragmented RNA can be used as input, however the template switching efficiency is lower for RNAs lacking a cap structure. Q: What type of RT primers can be used with the Template Switching RT Enzyme Mix? A: In general, the RT primers can be poly(dT), random hexamer or gene specific. For cDNA amplification, the RT primer should contain a PCR handle of known sequence for downstream cDNA amplification by PCR. Q: Can I reduce the RT reaction time? A: Reducing the RT reaction time is not recommended as it will likely decrease the yield of the desired cDNA product.