A total of 5 µL of RNA/DNA sample template was reverse transcribed using 1 µL random hexamers, 0.5 µL Oligo dT primer, 0.4 µL of dNTPs (10 mM) (Thermo Fisher Scientific, France) and 5.5 µL molecular grade water and incubated for 5 min at 65 °C. Next, 4 µL of Buffer 5×, 2 µL of 0.1 M DTT (M-MLV Reverse Transcriptase, Thermo Fisher Scientific, France) and 1 µL of RNAse OUT were added to the previous mix. After incubation for 2 min at 37 °C, 1 µL of M-MLV Reverse Transcriptase (M-MLV Reverse Transcriptase, Invitrogen, Thermo Fisher Scientific, Illkrich, France) was added to the mixture followed by incubation at 25 °C for 10 min, 37 °C for 50 min and 70 °C for 15 min. The cDNA obtained was stored at −20 °C.
M mlv reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China, Canada, Japan, France, United Kingdom, Lithuania, Netherlands, Switzerland, Italy, Finland, Argentina, Macao, Belgium, Austria
M-MLV reverse transcriptase is a recombinant enzyme used for the synthesis of first-strand cDNA from RNA templates. It catalyzes the conversion of single-stranded RNA into double-stranded cDNA.
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3 019 protocols using m mlv reverse transcriptase
1
Reverse Transcription of RNA/DNA Samples
2
Quantification of Sulfur Metabolism Genes in CT26 Cells
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CT26 cells were centrifuged 5 min at 400× g in order to resuspend the pellet in lysis buffer and isolate RNA using NucleoSpin RNA Plus kit (Macherey-Nagel, Düren, Germany). Nucleic acid concentration of samples was determined with NanoDrop spectrophotometer, and 3 µg of RNA was used for the reverse transcription using M-MLV Reverse Transcriptase (TermoFisher). RT-qPCR assays were performed with the following mouse primers purchased from Microsynth: CBS (Fw: 5′-CCT ATG AGG TGG AAG GGA TT-3′; Rev: 5′-TGT AGT TCC GCA CAG AGT CA-3′), 3-MST (Fw: 5′-ACA TCC CGG CTC AGT AAA CA-3′; Rev: 5′-TGT GTC CTT CAC AGG GTC TTC-3′), CSE (Fw: 5′-TGC CTC ACC CCA TTT CAT CT-3′; Rev: 5′-GAG TAA ACT GGG TGA GGG CT-3′), ETHE-1 (Fw: 5′-AGC TGC ACC TAT ACC TAC CTT C-3′; Rev: 5′-AGC TCC TTA ATC AAC TGA GCA TC-3′), TST (Fw: 5′-GGA GCC CGG ATA TAG TAG GAC TAG A-3′; Rev: 5′-TTC GTC AGG AAG TCC ATG AA-3′), and 18sRNA (Fw: 5′-GTA GTT CCG ACC ATA AAC GA-3′; Rev: 5′-TCA ATC TGT CAA TCC TGT CC-3′). SensiFAST Probe Hi-ROX Kit (Bioline, 82020, Memphis, TN, USA) was used for primers amplification with StepOnePlus Real-Time PCR System. The process was applied to four different passages of CT26 cell line.
3
Gene Expression Analysis of Plant Seedlings
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Total RNA was extracted from seedlings by using the Spectrum Plant Total RNA kit (Sigma). M-MLV reverse transcriptase (Thermal Fisher) was used to synthesize cDNA from the RNA. Quantitative real-time PCR was performed using LightCycler 480 (Roche) and the Bioline SYBR green master mix (Bioline). Gene expression levels were normalized to that of PP2A and are shown relative to the expression levels in wild type. Gene-specific primers are listed in Supplementary Table 1 .
4
Quantitative and Qualitative RT-PCR of Plant Genes
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For quantitative RT-PCR (qRT-PCR) assays, total RNA was extracted from the whole seedlings harvested under different time points. Two micrograms of total RNA was reversely transcribed in a 20 μl volume with M-MLV reverse transcriptase (Invitrogen). The fragments of target genes were amplified using SYBR Premix Ex Taq (TaKaRa). The thermal cycling program was 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s. ACTIN2 (At3G18780) was used for normalization the relative expression level of each transcript, and the comparative ΔΔCT method was employed to calculate the relative quantities of each amplified product. For identification of mutant alleles, total RNA was isolated from the leaves of 11-day-old plants grown in soil. Two micrograms of total RNA was reversely transcribed in a 20 μl volume using M-MLV reverse transcriptase (Invitrogen). The transcripts of specific genes were amplified with gene-specific primers. ACTIN2 was used as a reference. The primers used for qRT-PCR and semi-quantitative RT-PCR are listed in Table S1 .
5
DENV2 RNA Extraction and cDNA Synthesis
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The extraction of total RNA from mock- or DENV2-infected C6/36 cells using Isol-RNA Lysis Reagent (5PRIME Gaithersburg, MD, USA) followed the protocol provided by the manufacture. The extracted RNA was subsequently reversely transcribed to complementary DNA (cDNA) using the M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Briefly, the components including 4 μg of extracted RNA, 1 μL 100 mM Random hexamer primer (Fermentas, Glen Burnie, MD, USA) and 1 μL 10 mM dNTP Mix (Fermentas) were added into a 0.2-mL microcentrifuge tube, then filled with distilled water up to 12 μL of the total volume. The mixture was heated at 65 °C for 5 min, then 4 μL of 5× first-stand buffer, 2 μL 0.1 M DTT, 1 μL RNase OUTTM ribonucleases inhibitor (Invitrogen) and 1 μL of M-MLV Reverse Transcriptase were added. The final mixture was incubated at 37 °C for 60 min, followed by at 75 °C for 15 min. The formed cDNA was stored at −20 °C for further experiments.
6
Quantifying mRNA Levels in Microglia
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For tissue homogenates, 1 μg of RNA measured by spectrophotometry using Nanodrop (Thermo) was used for cDNA synthesis using M-MLV Reverse Transcriptase (Invitrogen). For isolated microglia from the hippocampus and striatum, the RNA yield was low, and its concentration was undetected by spectrophotometry or by a High Sensitivity mRNA Assay Kit (Q32852, Invitrogen); therefore, 8 μL of RNA samples was used for cDNA synthesis using M-MLV Reverse Transcriptase (Invitrogen). mRNA amplification was determined by RT-qPCR using SYBR green (Brilliant II SYBR green master mix, Agilent) and normalized to the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This strategy allowed for analyses of mRNA levels from purified microglia, and the threshold cycle of GAPDH was 26.7 ± 0.6 and 23.5 ± 0.8, for samples obtained from the hippocampus and striatum, respectively. Primer sequences are listed in Supplementary Table S1 .
7
Comprehensive miRNA and mRNA Expression Analysis
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Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen, Valencia, CA) and treated with RNase-Free DNase I (Qiagen),12 (link) quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific).
For quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis of miRNA expression, a poly(A) tail was first added to the 3′-end of miRNAs using a Poly(A) Polymerase Tailing Kit (Epicentre Biotechnologies, Madison, WI). Poly(A) tailed-miRNAs were then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen, Grand Island, NY) with a poly(T) adaptor, which consists of a poly(T) sequence and a sequence complementary to the universal primer used in following qRT-PCR analysis. SNORD44, SNORD47, and SNORD48 were used as internal controls. For qRT-PCR analysis of mRNA expression levels, total RNA was reversely transcribed using M-MLV Reverse Transcriptase with oligo(dT) 12–18 Primer (Invitrogen). Ribosomal protein L19 (RPL19) was used as internal control. qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on StepOnePlus or ViiA 7 Real-Time PCR System (Applied Biosystems). Primer sequences are in theTable S1 .
For quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis of miRNA expression, a poly(A) tail was first added to the 3′-end of miRNAs using a Poly(A) Polymerase Tailing Kit (Epicentre Biotechnologies, Madison, WI). Poly(A) tailed-miRNAs were then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen, Grand Island, NY) with a poly(T) adaptor, which consists of a poly(T) sequence and a sequence complementary to the universal primer used in following qRT-PCR analysis. SNORD44, SNORD47, and SNORD48 were used as internal controls. For qRT-PCR analysis of mRNA expression levels, total RNA was reversely transcribed using M-MLV Reverse Transcriptase with oligo(dT) 12–18 Primer (Invitrogen). Ribosomal protein L19 (RPL19) was used as internal control. qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on StepOnePlus or ViiA 7 Real-Time PCR System (Applied Biosystems). Primer sequences are in the
8
RNA Extraction and cDNA Synthesis for Viral Detection
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Total RNA was extracted and purified from cell culture supernatants or mosquito bodies using the NucleoSpin virus core kit (Macherey-Nagel) following the manufacturer’s instructions with RNA elution in 50 μl of RNase-free water at 70°C for bodies and 100 μl for cell culture supernatants. cDNA synthesis was performed using M-MLV reverse transcriptase (Invitrogen) by mixing 5 μl of eluted RNA with 100 ng of random primers (Roche), 10 nmol of each deoxynucleoside triphosphate, 2 μl of dithiothreitol, 4 μl of 5× first-strand buffer, 5.5 μl of PCR-grade water, 20 units of RNaseOUT recombinant RNase inhibitor (Invitrogen), and 200 units of M-MLV reverse transcriptase in a final reaction volume of 20 μl. The reaction mixtures were incubated for 10 min at 25°C, 50 min at 37°C, and 15 min at 70°C and held at 4°C until further use or stored at −20°C. Diagnostic PCRs for all three viruses was performed with DreamTaq Green DNA polymerase (Thermo Scientific) following the manufacturer’s recommendations. Quantitative analysis by qPCR was done using GoTaq qPCR master mix (Promega) following the manufacturer’s recommendations. The primer sequences are provided in Table 1 .
9
Mosquito Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted and purified from mosquito tissues using TRIzol Reagent (Invitrogen) following manufacturer’s instructions with RNA elution in 30 μL of PCR-grade water. cDNA synthesis was performed using M-MLV reverse transcriptase (Invitrogen) by mixing 10 μL of eluted RNA with 100 ng of random primers (Roche), 10 nmol of each dNTP, 2 μL of DTT, 4 μL of 5X First-Strand Buffer, 0.5 μL of PCR-grade water, 20 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 200 units of M-MLV reverse transcriptase in a final reaction volume of 20 μL. Reactions were incubated for 10 min at 25°C, 50 min at 37°C, 15 min at 70°C, and held at 4°C until further use or stored at −20°C. cDNA was diluted 1:5 before quantitative analysis by qPCR was done using GoTaq qPCR Master Mix (Promega) following manufacturer’s recommendations. Primer sequences are provided in Table S1 . CFAV qPCR values were normalized by the housekeeping gene rp49 qPCR values.
10
Extraction and Reverse Transcription of RNA
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Total RNA including miRNAs was extracted from ESCs using TRIzol (Invitrogen, USA). Revere transcription was performed on 2 ng of total RNA. Briefly, 2 ng of total RNA was reverse transcribed to cDNA. First strand cDNA of proto-oncogenes was synthesized by using M-MLV reverse transcriptase (Invitrogen, USA) with dT (18) oligo, and the First strand cDNA of miRNAs was synthesized by using M-MLV reverse transcriptase (Invitrogen, USA) with specific stem loop miRNA RT Primer (Ribo, China).
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