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Rapidxfire Thermostable Reverse Transcriptase Application Note Application note RapiDxFire Thermostable Reverse Transcriptase Introduction RNA target, which improves cDNA yield and A reverse transcriptase (RT) is a DNA subsequent detection sensitivity from difficult polymerase enzyme that synthesises cDNA RNA targets. Thermostability also is an from an RNA template. RTs are widely indicator of general enzyme stability for storage, used to study gene expression in cells or automation applications, and lyophilisation. tissues, in next-generation sequencing Processivity, which refers to the number of (NGS) applications, and in conjunction with nucleotides incorporated into cDNA during quantitative PCR (qPCR) or isothermal a single enzyme binding event, can affect amplification methods to detect and identify cDNA length, RT efficiency, and RT reaction RNAs that are of clinical or functional time. Native RT enzymes also have RNase H significance. endonuclease activity that will cleave the RNA from a DNA-RNA duplex and limit cDNA length. RT enzymes may differ in key characteristics Some RT variants also have been engineered that affect their performance in different to reduce RNase H activity to allow longer applications. Their thermostability allows cDNA products. cDNA synthesis to be performed at higher temperatures (above 50 °C), enabling them Commercially available RTs used in molecular to melt areas of secondary structure in the biology are generally derived from Moloney Application note RapiDxFire Thermostable Reverse Transcriptase murine leukaemia virus (MMLV) or avian Skeletal Muscle Total RNA (Thermo Fisher myeloblastosis virus (AMV), which have optimal Scientific Cat No. AM7982), RNA, MS2 (Roche activity at 37-42 °C. Cloned AMV RT and Cat No. 10165948001), Zika virus ATCC® engineered variants of AMV RT and MMLV VR-1843™ (ATCC Cat No. VR-1843), poly RT were developed to improve thermostability (rC)-p(dG) 12-18 (Midland Certified Reagents, and other performance characteristics. Newer Cat No. P-4210), and poly (rA)-p(dT) 12-18 applications, and use of difficult sample (Midland Certified Reagents, Cat No. P-4012). types and targets, require RTs that are not only thermostable but also demonstrate high Activity assay sensitivity and shorter reaction times. We Activity profiles were generated using the have found that most commercially available Invitrogen™ EnzChek™ Reverse Transcriptase engineered RTs have limited activity at, and Assay Kit (Thermo Fisher Scientific Cat No. tolerance to, higher temperatures, and suffer E22064) utilising either poly(A) template limited stability under ambient temperature or poly(C) template. The RNA-DNA conditions and in use with automated handling heteroduplexes were then quantified utilising platforms. PicoGreen® fluorescence assay on a Tecan Infinite® M1000 Pro microplate reader. RapiDxFire™ Thermostable Reverse Transcriptase is a unique, proprietary enzyme cDNA synthesis originally identified from a hot spring viral RapiDxFire Thermostable Reverse source. The RapiDxFire Thermostable RT Transcriptase (Lucigen Cat No. 30250-2) was retains its inherent stability under multiple used according to the standard protocol. RTs storage and temperature conditions, and from other suppliers were used according provides strong reverse transcriptase activity to their standard protocols unless otherwise at high temperatures. This enzyme, which stated. For all first-strand cDNA synthesis lacks RNase H and 3ʹ→ 5ʹ exonuclease steps, gene-specific primers were utilised. activity, efficiently synthesises short cDNA fragments (≤ 1 Kb). Like other RTs, RapiDxFire PCR RT possesses DNA polymerase activity but PCR was performed on samples in a second lacks 5ʹ→ 3ʹ exonuclease activity. RapiDxFire step following reverse transcription using Thermostable Reverse Transcriptase performs EconoTaq DNA Polymerase (with Mg++) fast and efficient first-strand cDNA synthesis and its supplied buffer (Lucigen Cat No. using gene-specific primers. In this study, we 30031-3). Real-time detection of PCR products compared RapiDxFire Thermostable Reverse was performed using an intercalating dye Transcriptase thermostability, speed, and (Dyomics Cat No. V13-01184) and a BioRad sensitivity to two leading thermostable RTs in CFX C1000 Touch™ Thermal Cycler with the market. maximum absorption/emission wavelengths of 481nm/526nm, respectively. The reverse Materials and Methods transcription reaction contributed up to 10% of Commercial samples the total PCR assay volume. Commercial samples used included Human Application note RapiDxFire Thermostable Reverse Transcriptase RapiDxFire Thermostable RT Supplier A Supplier Activity Activity Activity 37 50 55 0 5 70 75 0 37 50 55 0 5 70 75 0 37 50 55 0 5 70 75 0 Reaction temperature (°C) Reaction temperature (°C) Reaction temperature (°C) Figure 1. Reaction temperature profiles for thermostable reverse transcriptases. Triplicate reactions were set up on ice for each condition tested using each manufacturer’s recommended buffer system, transferred to the indicated temperatures, and incubated for 40 minutes. RNA/cDNA RNA- DNA heteroduplexes were then detected utilising PicoGreen fluorescence assays on a Tecan Infinite M1000 Pro microplate reader. Results Time course and sensitivity Reverse transcription activity thermal Time-course studies of cDNA synthesis were profile performed in a 2-step RT-qPCR process Thermal activity profiles were determined for with different thermostable RTs. Zika cDNA each enzyme by setting up triplicate reactions synthesis was conducted for each enzyme in on ice for each condition tested using each duplicate using target-specific primers in the manufacturer’s recommended buffer system recommended reaction buffer and incubation and then incubating the RT reactions with temperatures (RapiDxFire: 60 °C; Supplier poly (rC)-p(dG) 12-18 template/primer for A: 50 °C; and Supplier B: 55 °C) according to 40 minutes at eight different temperatures (37, the suppliers’ guidelines. After completion of 50, 55, 60, 65, 70, 75, and 80 °C). Following synthesis and a 1-minute reverse transcription incubation, RNA/cDNA product was quantified reaction (Figure 2A), qPCR was performed by PicoGreen dye fluorescence as a measure using one-tenth volume of each of the Zika of polymerisation activity. Each profile was cDNA samples using an intercalating dye. represented as a percentage of each peak Additional samples were run through 1-, 2-, 5-, RFU (and not as a comparison against each and 10-minute reverse transcription reactions other). RapiDxFire Thermostable RT exhibited for a time-course study prior to performing increasing activity as the reaction temperature second-step qPCR. Encircled data points are was increased up to 80 °C, the highest derived from data represented in the qPCR temperature tested, and retained ~60% activity curve from panel A (Figure 2). after 10 minutes at 90 °C (data not shown). The reverse transcriptases from Supplier B and Supplier A had peak activities between 50-55 °C and 37-50 °C, respectively (Figure 1). Application note RapiDxFire Thermostable Reverse Transcriptase A. -minute RT reaction . -, 2-,A. 5-, and 0-minuteika RT virus reactions . eta actin gene (total RNA) RapiDxFire Supplier A Supplier RapiDxFire RapiDxFireSupplier A SupplierSupplier A Supplier RapiDxFire Supplier A Supplier ,000 45 45 45 40 ,000 40 40 35 35 4,000 35 RFUs Cq value Cq value Cq value 30 30 2,000 30 25 25 0 25 55 °C 60 °C 65 °C 55 °C 0 °C 5 °C 20 25 30 35 40 1 2 3 4 5 6 7 8 9 0 Reaction temperature Reaction temperature Cycles Time (minutes) A. -minute RT reaction . -, 2-,A. 5-, and 0-minuteika RT virus reactions . eta actin gene (total RNA) RapiDxFire Supplier A Supplier RapiDxFire Supplier A Supplier RapiDxFire Supplier A Supplier RapiDxFire Supplier A Supplier 45 ,000 45 45 40 40 ,000 40 35 35 4,000 35 RFUs Cq value Cq value 30 30 Cq value 2,000 30 25 25 55 °C 60 °C 65 °C 55 °C 0 °C 5 °C 0 25 20 25 30 35 40 1 2 3 4 5 Reaction6 7 temperature8 9 0 Reaction temperature Cycles Time (minutes) Figure 3. Reaction temperature sensitivity. Real-time qPCR Figure 2. cDNA synthesis time course studies in a 2-step RT-qPCR detection after a two-step real-time RT-qPCR process that created process with different thermostable RTs. Zika cDNA synthesis for cDNA from (A) Zika virus or (B) beta actin gene from total Human each enzyme performed in the recommended reaction buffer and RNA using different RT reaction temperatures of 55, 60, and 65 °C incubation temperature recommended for RapiDxFire, Supplier for 10 minutes. A, and Supplier B. After synthesis and RT reaction, qPCR was performed using one-tenth volume of each of the cDNA samples using an intercalating dye in 1-minute (A) or (B) 1-, 2-, 5-, and separate reactions each enzyme was used 10-minute RT reactions. Data points derived from the qPCR curve in to create cDNA from Zika virus (Figure 3A) panel A are encircled in panel B. or using 0.5 ng of Human total RNA (Figure 3B) using RT reaction temperatures of 55, Reaction temperature impact on sensitivity 60, and 65 °C for 10 minutes. After cDNA Reaction temperature and sensitivity were synthesis, real-time qPCR was run on each evaluated using a two-step real-time RT-qPCR of the RT samples. RapiDxFire Thermostable process. cDNA synthesis was conducted for RT provided consistent results across reaction each of the enzymes using standard conditions temperatures with superior sensitivity. according to each supplier’s guidelines. In Application note RapiDxFire Thermostable Reverse Transcriptase Thermostability RapiDxFire Supplier A Supplier 55 °C 0 °C 5 °C 20 20 20 00 00 00 0 0 0 0 0 0 40 activity activity 40 activity 40 20 20 20 0 0 0 0 5 30 45 0 0 5 30 45 0 0 5 30 45 0 Preincubation time (minutes) Preincubation time (minutes) Preincubation time (minutes) Figure 4. Thermostability studies of RTs at elevated temperatures. Reaction mixes containing each enzyme, but without dTTP and poly (rA)-p(dT) 12-18 template/primer were pre-incubated for 0 to 60 minutes at 55, 60, and 65 °C. After adding nucleotide and template/primer, 15-minute RT reactions at 55 °C were performed before quantifying the RNA/cDNA product by PicoGreen fluorescence.
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