Here we provide a complete protocol to standardize and implement the method for detecting the SARS-CoV-2 virus in human samples by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method, done within 60 min, could be adapted to any laboratory or point-of-care at a low cost and using inexpensive equipment.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has dramatically impacted human health. It continues to be a threat to modern society because many people die as a result of infection. The disease is diagnosed using serologic and molecular tests, such as the gold standard real-time polymerase chain reaction (RT-PCR). The last has several disadvantages because it requires specialized infrastructure, costly equipment, and trained personnel. Here, we present a protocol outlining the steps required to detect the SARS-CoV-2 virus using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) in human samples. The protocol includes instructions for designing primers in silico, preparing reagents, amplification, and visualization. Once standardized, this method can be easily implemented and adapted to any laboratory or point-of-care within 60 min at a low cost and using inexpensive equipment. It is adaptable to detecting different pathogens. Thus, it can potentially be used in the field and in health centers to carry out timely epidemiological surveillance.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The World Health Organization declared a public health emergency of international concern on 30 January 2020 and a pandemic on 11 March 2020. The pandemic resulted in over 760 million cases and 6.87 million deaths as of the date this article was written1.
The impact of this virus has highlighted the need for better, more accurate, faster, and more widely available surveillance tools to improve infectious disease detection and control2,3. During the pandemic, SARS-CoV-2 diagnostic tests were based on detecting nucleic acid, antibodies, and proteins, but RT-PCR detection of nucleic acid is the gold standard4. However, RT-PCR has some limitations; it requires specialized equipment, infrastructure, and personnel trained in molecular biology, limiting its application to specialized laboratories. Further, it is time-consuming (4-6 h), not including the time to transport the specimens to the laboratory, which can take days5. These constraints prevent efficient sample processing and obtaining the information required for contingency planning and epidemiological management.
Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) has several advantages over RT-PCR, making it an appealing strategy for designing future point-of-care diagnostic tests (POCT), particularly in resource-constrained settings6. First, it is greatly specific because it uses between four and six primers that recognize six to eight areas in the target sequence, be it DNA or RNA7,8. Second, because it operates at a constant temperature, it does not require sophisticated equipment such as real-time thermal cyclers to generate the amplification, nor does it necessitate highly trained personnel to operate it. Third, the reaction time is very short (~60 min), and reagents that are not very specialized are employed, which makes it a cost-effective tool6. Given the foregoing and the health emergency caused by the COVID-19 pandemic, this technique can be viewed as an alternative diagnostic method that is quick, inexpensive, and simple to implement in any research laboratory9.
The protocol for standardizing and implementing an RT-LAMP to detect SARS-CoV-2 by colorimetric methods using a thermocycler and a water bath is described in this article (Figure 1). Critical points, their limitations, and alternatives to advance them are discussed.
Figure 1: Scheme of the protocol for amplifying SARS-CoV-2 using the RT-LAMP technique. Please click here to view a larger version of this figure.
The samples used were provided by the clinical laboratory of Fundación Valle del Lili University hospital and corresponded to the purified RNA from patients who tested positive for COVID-19 using the RT-qPCR technique. All patients provided informed consent for research, and this study was approved by the bioethical committee for human studies from Fundación Valle del Lili University hospital.
1. RT-LAMP primer design and preparation
NOTE: LAMP primers can be used with a variety of platforms, including New England BioLabs (NEB) LAMP, Primer Explorer, and LAMP assay versatile analysis (LAVA). However, for this protocol, the NEB LAMP tool was used. Primer design can be done using SARS-CoV-2 genomes obtained from NextStrain database10. Table 1 shows the primer set used in this protocol.
2. RT-LAMP reaction
3. Analysis of amplification products in agarose gel
NOTE: These steps are suggested as additional checks to colorimetric reaction or control for performance during the standardization step. This is because the technique could present a huge contamination risk to the lab doing these tests.
The implementation of the protocol starts by designing the set of primers for each target gene following the protocol described above. In June 2020, 5,000 SARS-CoV-2 genomes were obtained from the NextStrain database, with a 10% representativeness of Colombian genomes. These sequences were aligned to obtain the consensus sequence that was used in the primer design process. Table 1 shows the primers set chosen for primers RdRp/Hel and RdRp. The primer set for gene N amplification was obtained from a previously published report14.
The first step in the standardization of the protocol was to avoid NTC amplification. One of the most important parameters that must be verified in this regard is determining the optimal melting temperature (Tm) and Bst 3.0 concentration for the set of primers. A temperature gradient was used to determine the best Tm for the amplification (Figure 4A). For the set of primers used in this protocol, the optimal Tm was 66.3 °C (Figure 4A). Furthermore, different concentrations of the Bst 3.0 enzyme were evaluated, with 3.2 lU/µL being the optimal concentration for that reaction (Figure 4B). The concentrations provided by Lu et al.15 were used for the remaining reagents (Table 3, Table 4, and Table 5).
Once the Tm and Bst 3.0 optimal concentration was determined, the amplification process was carried out. The patient samples were provided by University Hospital Fundación Valle del Lili. The positive and negative samples were previously amplified using a conventional RT-PCR protocol, and the viral RNA was then used for RT-LAMP amplification using the protocol described here and the conditions listed. The amplification of N, RdRp/Hel, and RdRp genes in the patient samples but not in NTC is shown in Figure 5A,B. Given that the RT-LAMP is an appealing strategy for designing POCT in the future, the amplifications in this protocol were implemented in a conventional thermal cycler and a water bath (Figure 6).
The second step in standardizing the protocol was to define the colorimetric strategy, so phenol red and neutral red were the first dyes evaluated; for its evaluation, different dyes concentration was probed (Figure 7), but no color change was observed after amplification with any of the concentrations tested. This result could be explained by the fact that both dyes are pH indicators, meaning they are sensitive to the pH of the sample, especially if the viral RNA was eluted in Buffer TE, which was the case for the patient samples evaluated with this protocol. Hydroxy Naphthol Blue (HNB) was investigated because its color changes with changes in Mg2+ concentration, which would be reduced during the amplification reaction as a cofactor of the polymerase enzyme. In this case, different concentrations of HNB were tested to determine which allowed for the best color change after amplification, which occurs in the reaction with 125 µM of HNB (Figure 8). A complete description of reagents employed in the preparation of the amplification mix for N and RdRp/Hel genes by colorimetric LAMP is included in Table 5.
After determining the best conditions for colorimetric detection, patient samples with varying numbers of viral genomes were amplified. Figure 9 shows the amplification of the samples, in which a change of color occurs in the samples according to the concentration of the viral genome. The amplification results were also visualized using agarose electrophoresis, confirming the amplification of patient samples but not NTC.
Figure 2: Diagram of the water bath used to amplify SARS-CoV-2 genes using the LAMP technique. (A) Front view and (B) top view. Please click here to view a larger version of this figure.
Figure 3: Electrophoresis chamber assembly. (A) Diagram of the electrophoresis chamber used to separate the PCR products. (B) Formation of the internal chamber for the preparation of the agarose gel. (C) Addition of the molten agarose in the internal chamber. (D) Disassembly of the inner chamber to accommodate the gel in the running position. Please click here to view a larger version of this figure.
Figure 4: Standardization of the amplification conditions of the primers and the enzyme Bst 3.0. (A) Each lane shows a different gradient temperature, ranging from 63 °C to 67 °C. From left to right, molecular weight marker (MWM), No Template Control (-), lane 1: 67 °C; lane 2: 66.8 °C; lane 3: 66.3 °C; lane 4: 65.5 °C; lane 5: 64.6 °C; lane 6: 63.9 °C; lane 7: 63.4 °C; lane 8: 63 °C. (B) B1: 8.0 U/µL of Bst 3.0; B2: 6.4 U/µL of Bst 3.0; B3: 4.8 U/µL of Bst 3.0; B4: 3.2 U/µL of Bst 3.0; 1: No Template Control; and 2: patient sample EEDD8 (Ct = 23.39). Please click here to view a larger version of this figure.
Figure 5: Agarose gel electrophoresis of the amplification products of the N and RdRp/Hel genes present in the SARS-CoV-2 virus using the LAMP technique. (A) Replicate 1 and (B) Replicate 2, where MWM: molecular weight marker; 1: No Template Control; 2: patient sample E1123 (Ct = 19.95); 3: patient sample E1324 (Ct = 26.01); 4: patient sample EEDD10 (Ct = 30.09); RH: RdRp/Hel gene and N: N gene. Please click here to view a larger version of this figure.
Figure 6: Agarose gel electrophoresis of the amplification products of the RdRp gene present in the SARS-CoV-2 virus using the LAMP technique. The reaction was carried out in (A) a thermal cycler and (B) a water bath system. MPM: molecular weight marker; 1: No Template Control; 2: patient sample E1123 (Ct = 19.95); 3: patient sample E1757 (Ct = 23.67); 4: patient sample E1604 (Ct = 23.98); 5: patient sample E1245 (Ct = 25.99); 6: patient sample E1324 (Ct = 26.01); 7: patient sample EEDD7 (Ct = 26.56); 8: patient sample 24 (Ct = 37.99) and R: RdRp gene. *Refers to the amplifications that were subjected to 60 min of reaction. Please click here to view a larger version of this figure.
Figure 7: Amplification of the RdRp gene present in the SARS-CoV-2 virus using the LAMP technique with colorimetric detection. Tubes (A) before and (B) after the reaction, where 1: No Template Control; 2: patient sample E1123 (Ct=19.95); 3: patient sample E1757 (Ct=23.67); 4: patient sample E1324 (Ct=26.01); PR: Phenol Red; and NR: Neutral Red. For each dye, four concentrations were evaluated in the final reaction (50 µM, 75 µM, 100 µM, and 120 µM). Please click here to view a larger version of this figure.
Figure 8: Amplification of the RdRp gene present in the SARS-CoV-2 virus by the colorimetric LAMP technique, using the blue hydroxynaphthol indicator. Tubes (A) before and (B) after the reaction, where 1: No Template Control; 2: patient sample E1594 (Ct=20.75); 3: patient sample E990 (Ct=22.67); 4: patient sample E1245 (Ct=25.99). For this dye, four concentrations were evaluated in the final reaction (50 µM, 75 µM, 100 µM, and 125 µM). Please click here to view a larger version of this figure.
Figure 9: Amplification of the samples. Tubes (A) before and (B) after reaction by LAMP using the blue hydroxynaphthol indicator. (C) Agarose gel electrophoresis of the amplification products of the N gene present in the SARS-CoV-2 virus using the colorimetric LAMP technique. MPM: molecular weight marker; NTC: No Template Control; S1: patient sample E1594 (Ct = 20.75); S2: patient sample E990 (Ct = 22.67); S3: patient sample E1245 (Ct = 25.99). Please click here to view a larger version of this figure.
Target Gene | Primer | Primer Sequence (5’ → 3’) | |||
N (Zhang et al., 2020) |
N-F3 | TGGCTACTACCGAAGAGCT | |||
N-B3 | TGCAGCATTGTTAGCAGGAT | ||||
N-FIP | TCTGGCCCAGTTCCTAGGTAGTCCAGACGAATTCGTGGTGG | ||||
N-BIP | AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT | ||||
N-LF | GGACTGAGATCTTTCATTTTACCGT | ||||
N-LB | ACTGAGGGAGCCTTGAATACA | ||||
RdRp | R-F3 | CTATGGTGGTTGGCACAA | |||
R-B3 | TTGAGCACACTCATTAGCT | ||||
R-FIP | GCATGGCTCTATCACATTTAGGATA-GTTTATAGTGATGTAGAAAACCCTC | ||||
R-BIP | ACATGCTTAGAATTATGGCCTCAC-TCTATAGAAACGGTGTGACAAG | ||||
R-LB | TGTTCTTGCTCGCAAACATACAACG | ||||
RdRp/Hel | RH-F3 | GGTATTGGGAACCTGAGTT | |||
RH-B3 | GACAAGACTAATTTATGTGATGTTG | ||||
RH-FIP | GCAAAGAACACAAGCCCCAACTTATGAGGCTATGTACACACC | ||||
RH-BIP | TTCACAGACTTCATTAAGATGTGGTACATGGTCGTAACAGCAT | ||||
RH-LB | GCTTGCATACGTAGACCATTCTT |
Table 1: Primer sequences for SARS-CoV-2 detection by RT-LAMP.
Component | Stock (µM) | Primer Mix 10x (µM) | Volume (µL) |
Forward Outer Primer (F3) | 100 | 2 | 12.5 |
Backward Outer Primer (B3) | 100 | 2 | 12.5 |
Forward Inner Primer (FIP) | 100 | 8 | 50 |
Backward Inner Primer (BIP) | 100 | 8 | 50 |
Loop Forward (LF) | 100 | 4 | 25 |
Loop Backward (LB) | 100 | 4 | 25 |
Nuclease-free Water* | — | — | 450 |
Total Volume | 625 |
Table 2: Preparation of 10x RT-LAMP primer mix. The RdRp gene primer mix does not contain the LF primer; therefore, replace this volume with Nuclease-free water. *Instead of Nuclease-free water, 10 mM Tris pH 8.0 prepared in DEPC 0.1% water can be used.
Item | Reagents | Final concentration for 25 μL | 1 sample (μL) |
1 | 10x Buffer | 1x | 2.5 |
2 | 100 mM MgSO4 | 4 mM + 2 mM in buffer = 6 mM | 1.0 |
3 | 10 mM dNTPs | 1.4 mM | 3.5 |
4 | 10x Mix Primers | 1x [ 0.2 μM F3/B3; 0.8 μM FIP/BIP; 0.4 μM LB] | 2.5 |
5 | Bst 3.0 DNA pol (8000 IU/mL) | 3.2 IU | 0.4 |
6 | RTx (15000 IU/mL) | 1.5 IU | 0.1 |
7 | Q5 DNA pol (2000 IU/mL) | 0.15 IU | 0.1 |
8 | Nuclease-free Water | N/A | 11.9 |
9 | RNA sample | N/A | 3.0 |
10 | Total Reaction Volume | 25 |
Table 3: Preparation of the RdRp gene amplification mix by LAMP.
Item | Reagents | Final concentration for 25 μL | 1 sample (μL) |
1 | 10x Buffer | 1x | 2.5 |
2 | 100 mM MgSO4 | 6 mM + 2 mM in buffer = 8 mM | 1.5 |
3 | 10 mM dNTPs | 1.4 mM | 3.5 |
4 | 10x Mix Primers | 1x [ 0.2 μM F3/B3; 0.8 μM FIP/BIP; 0.4 μM LF/LB] | 2.5 |
5 | Bst 3.0 DNA pol (8000 IU/mL) | 3.2 IU | 0.4 |
6 | RTx (15000 IU/mL) | 1.5 IU | 0.1 |
7 | Q5 DNA pol (2000 IU/mL) | 0.15 IU | 0.1 |
8 | Nuclease-free Water | N/A | 11.4 |
9 | RNA sample | N/A | 3.0 |
10 | Total Reaction Volume | 25 |
Table 4: Preparation of the amplification mix of the N-A and RdRp/Hel genes by LAMP.
Item | Reagents | Final concentration for 25 μL | 1 sample (μL) |
1 | 10x Buffer | 1x | 2.5 |
2 | 100 mM MgSO4 | 6.5 mM + 2 mM in buffer = 8.5 mM | 1.6 |
3 | 10 mM dNTPs | 1.4 mM | 3.5 |
4 | 10x Mix Primers | 1x [ 0.2 μM F3/B3; 0.8 μM FIP/BIP; 0.4 μM LF/LB] | 2.5 |
5 | 1 mM Hydroxy naphthol blue | 125 μM | 3.1 |
6 | Bst 3.0 DNA pol (8000 IU/mL) | 3.2 IU | 0.4 |
7 | RTx (15000 IU/mL) | 1.5 IU | 0.1 |
8 | Q5 DNA pol (2000 IU/mL) | 0.15 IU | 0.1 |
9 | Nuclease-free Water | N/A | 8.2 |
10 | RNA sample | N/A | 3.0 |
11 | Total Reaction Volume | 25 |
Table 5: Preparation of the amplification mix of the N-A and RdRp/Hel genes by colorimetric LAMP.
Temperature | Time |
66.3 °C | 60 min |
80 °C | 5 min |
4 °C | ∞ |
Table 6: Thermal conditions used for the amplification of the RdRp, N-A, and RdRp/Hel genes by LAMP.
Dye | pH Dependent | Color Before Reaction | Color After Reaction | Comments | ||
Hydroxy naphthol blue | No | Violet | Sky Blue | With this dye the magnesium concentration is critical and must be between 8 mM and 8.5 mM in the final reaction. In this way, the color transition from violet to sky blue is guaranteed. | ||
Cresol red | Yes | Fuscia | Yellow | The presence of buffers in the RNA eluates or in the reagents can prevent the reduction of pH and affect the color change when the reaction is finished. Therefore, in the case of RNA and primers, it is recommended not to use TE buffer for elution and resuspension/dilution, respectively. | ||
Neutral Red | Yes | Faint yellow or Faint orange | Fuscia | |||
Phenol Red | Yes | Fuscia | Yellow |
Table 7: Comparison of the dyes used in the visual detection of colorimetric LAMP.
Although the RT-LAMP is regarded as a complementary methodology for performing molecular diagnostics, it also has some limitations and critical steps that must be considered when the protocol is standardized and implemented.
The LAMP standardization for the detection of SARS-CoV-2 evaluated the following parameters and components in the master mix: (a) concentration and temperature of alignment of the primers; (b) concentration of the enzymes; (c) magnesium concentration; (d) reaction time; (e) inclusion of additives such as BSA, DMSO, Guanidinium Chloride; (f) design of the primers; (g) addition of colorant; (h) use of reaction buffers and recombinant enzymes produced in-house.
This standardization process began with six sets of primers, two reported by Zhang et al.14 and four designed by the BioDx company. The latter was designed from scratch with available online tools and was selected for its high specificity against SARS-CoV-2, as determined by BLAST. In addition, their thermodynamic evaluation showed a lower tendency to form primer dimers or hairpin structures that favored self-amplification.
Due to the impact that primers have on the standardization of the technique and the results of previous experiments where amplification was found in NTCs, a new primer design was made for the N, S, RdRp, and RdRp/Hel genes. These were subjected to several bioinformatic analyzes to guarantee their specificity and the reduction of the tendency to form self-amplifying structures. The selected primers met the established bioinformatics parameters (ΔG > -5.0 and specificity demonstrated by BLAST).
In silico assays have a very important role in predicting the behavior that can be observed in in vitro reactions since they can be used as an initial filter for the selection of primers. However, these predictions are not the same in all cases; therefore, they cannot be used as a definitive selection parameter but rather as an initial approximation. The actual behavior of the primers in the technique is determined directly during the reaction and visual evaluation by colorimetry or on an agarose gel. Finally, doubtful or unclear results are usually repeated by another analyst or discarded; since an inconclusive result cannot be reported in the context of the diagnosis.
One of the most important critical steps is the high probability of contamination when the protocol is being developed, which is reflected in the amplification of the NTC. This could be avoided by following good laboratory practices such as proper disinfection and cleaning procedures before handling reagents and samples, always wearing the required PPE, handling implements with care inside the laminar flow cabinet, and developing each step of the process in separate spaces for exclusive use, such as a space to mix reagents, another to add the sample to be evaluated and another to carry out the amplification process.
The NTC amplification could be caused by contamination but also by primer dimerization. For this reason, another critical step is the primer design and identifying the optimal conditions to prevent primer dimerization. It is critical in the primer design process to find the primer set with thermodynamic parameters that reduce the likelihood of secondary structures forming, which is measured with free energy, so a thermodynamic evaluation of the primer set chosen is required. In this protocol, the PrimerDimer tool12 was employed for the thermodynamic evaluation; with the Multiplex examination option, each primer is screened against all other primers within the pool for potential dimer formations, and the Dimer Framework Report option reports the framework of the most stable dimer of each primer pair. This information is very helpful in selecting a good set of primers.
Furthermore, identifying the appropriate reagent concentration such as MgSO4, dNTPs, primers, Bst 3.0 enzyme, and amplification parameters such as Tm and incubation time is critical to preventing primer dimerization during the amplification process. Besides, it is possible to add compounds that reduce the formation of primer dimers during amplification, for example, bovine serum albumin, dimethyl sulfoxide (DMSO)16, and guanidinium chloride17.
Another critical aspect of the primer design process is determining the specificity of each primer. For this protocol, this evaluation was made with BLAST. However, it is also important to evaluate the specificity of the primers amplification process using another virus that is phylogenetically related but not related to SARS-CoV-2.
On the other hand, the sensitivity and the detection limit (minimum number of detectable genomes) were not determined during the standardization of the technique. In the first place, the samples delivered by University Hospital Fundación Valle del Lili were not quantified to determine the viral copies number due to internal restrictions derived from the sanitary contingency that was in place at that time in Colombia (2020). The estimate of the concentration of genetic material was approximated with the Cq of the RT-qPCR since the lower the Cq, the higher the viral copies number, while the higher the Cq, the lower the viral copies number. The lowest Cq tested was 14.81, and the highest was 38.93, where no amplification products appear. Additionally, in 2020 the RT-qPCR test approved for use in Colombia was qualitative and not quantitative, that is to say, only determined presence or absence according to Cq. A result with Cq ≥ 40 was negative, but a Cq < 40 was positive. The protocols used in Colombia for the detection of COVID-19 were the Berlin protocol1, the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR diagnostic Panel2, and particularly in University Hospital Fundación Valle del Lili, the Allplex 2019-nCoV Assay3 was used. Unfortunately, we do not have any more samples approved by the ethics committee supplied by University Hospital Fundación Valle del Lili to continue with these experiments.
Additionally, the availability of reagents is a significant barrier to the timely diagnosis of new cases in a global public health emergency the one caused by SARS-CoV-2. As a result, this protocol was created to avoid the use of imported and costly commercial kits and reagents, and the preparation of the amplification buffers is detailed in this protocol. Furthermore, some dyes that could be used in colorimetry assays were evaluated, as well as some considerations for their use (Table 7).
This diagnostic method has some limitations because it does not allow for the quantification of the viral genome in the sample. In addition, color detection is a subjective measure because it depends on the visual capacity of the person performing the protocol. However, because it does not require specialized equipment or personnel trained in molecular biology, this protocol is adaptable to the detection of different pathogens and could be easily implemented once it has been standardized. Thus, its application could be extended to other samples, such as environmental18,19,20 and health centers, to carry out timely epidemiological surveillance.
The authors have nothing to disclose.
This work was funded by Sistema General de Regalías of Colombia, grant number BPIN 2020000100092, and Universidad Icesi – Convocatoria Interna, grant number CA0413119. MFVT was also financed by the Assistant Professorship Funds from Universidad de los Andes. The funding entities did not participate in the design, execution of activities, data collection, and data analysis and preparation of the manuscript. We thank to University Hospital Fundación Valle del Lili for viral RNA from Sars -CoV-2 samples and Dr. Alvaro Barrera-Ocampo for the comments on the manuscript.
1 kb DNA Ladder | SOLIS BIODYNE | 07-12-00050 | Store at -20 °C |
50x TAE Electrophoresis Buffer | ThermoScientific | B49 | Store at roome temperature |
Accuris High Fidelity Polymerase | ACCURIS LIFE SCIENCE REAGENTS | PR1000-HF-200 | It can be used in case Q5 High-Fidelity DNA polymerase cannot be purchased. For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
Agarose | PanReacAppliChem | A8963,0100 | N/A |
Bst 3.0 DNA Polymerase 8000 IU/mL | New England BioLabs | M0374S/M0374L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
Deoxynucleotide (dNTP) Solution Set | New England BioLabs | N0446S | Store at -20 °C |
Diethyl pyrocarbonate | Sigma-Aldrich | 159220-25G | Handle it with caution under an extraction cabinet |
GeneRuler 100 bp Plus DNA Ladder, ready-to-use | ThermoScientific | SM0322 | Store at -20 °C |
Hydroxy naphthol blue disodium salt | Santa Cruz Biotechnology | sc-215156B | N/A |
Q5 High-Fidelity DNA polymerase 2000 IU/mL | New England BioLabs | M0491S/M0491L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |
WarmStart RTx Reverse Transcriptase 15000 IU/mL | New England BioLabs | M0380S/M0380L | For the enzyme, make aliquots of an appropriate volume and store at -20 °C until use. |