cDNA was prepared by reverse transcription using total RNA as the template, and 1 μg of each RNA sample heat-treated at 65 °C was mixed with 20% five First Strand Buffer (Thermo Fisher Scientific), 1 mM dithiothreitol (Life Technologies, Gaithersburg, MD, USA), and 1 μg of each RNA sample heat-treated at 65 °C. The final concentration shown below was added to the sample: (Gaithersburg, MD, USA), 1.1-U/µL ribonuclease inhibitor (Takara Bio, Shiga, Japan), 0.5-mM dNTP mixture (Takara Bio), 5-U/µL Moloney-Mouse leukemia virus reverse transcriptase (Life technologies), and 55 ng/µL random Hexamers (Pharmacia Biotech, Milwaukee, WI, USA). The reaction solution was stored at 37 °C for 60 min and subsequently treated at 99 °C for 5 min to inactivate the residual enzymes for cDNA preparation.
Mouse moloney leukemia virus reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States
Mouse Moloney leukemia virus reverse transcriptase is an enzyme that catalyzes the conversion of single-stranded RNA into double-stranded complementary DNA (cDNA). This enzyme plays a crucial role in the reverse transcription process, which is a fundamental step in various molecular biology techniques, such as gene expression analysis and viral genome detection.
Automatically generated - may contain errors
Lab products found in correlation
Ribonuclease inhibitor, by Takara Bio (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
First strand buffer, by Thermo Fisher Scientific (1 mentions)
Dntp mixture, by Takara Bio (1 mentions)
Random hexamers, by GE Healthcare (1 mentions)
Cfx96 qpcr system, by Bio-Rad (1 mentions)
Gotaq master mix, by Promega (1 mentions)
Lightcycler 480 sealing foil, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Deoxynucleoside triphosphate, by Promega (1 mentions)
Thermocycler, by Thermo Fisher Scientific (1 mentions)
Rneasy minikit procedure, by Qiagen (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Veriti pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Image analysis system, by Bio-Rad (1 mentions)
5 protocols using mouse moloney leukemia virus reverse transcriptase
1
Reverse Transcription of Total RNA
2
Quantitative PCR for Zika Virus RNA
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Total RNA including genomic viral RNA was extracted from cells (Qiagen, Hilden, Germany) and reverse transcription was performed using 500 ng of total RNA, random hexamer primers (intracellular viral RNA) or E reverse primer (virus particles) and moloney mouse leukemia virus reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 °C for 50 min. Quantitative PCR was performed on a CFX96 qPCR System (Bio-Rad, Hercules, CA, USA). Briefly, 10 ng of cDNA was amplified using 0.2 μM of each primer and 1X GoTaq Master Mix (Promega, Madison, WI, USA). When appropriate, data were normalised to the internal standard GAPDH. For each single-well amplification reaction, a threshold cycle (Ct) was calculated using the CFX96 qPCR program (Bio Rad, Hercules, CA, USA) in the exponential phase of amplification. Relative changes in gene expression were determined using the 2ΔΔCt method and reported relative to the control. Primers used in this study are listed in [31 (link)]. ZIKV E primers were designed to match both MR766-NIID and BeH819015 sequences (forward 5-gtcttggaacatggagg-3′ and reverse 5′-ttcaccttgtgttgggc-3′).
3
Quantitative RT-PCR Protocol for Gene Expression
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cDNA was synthesized using mouse Moloney leukemia virus reverse transcriptase (Invitrogen) according to manufacturer's instructions. For reverse transcription, 0.3 µg aliquots of total RNA were used from the same samples, which were used for microarray analysis. qPCRs were performed using a PCR mastermix supplemented with 0.2 M trehalose, 1 M 1,2-propanediol and SYBR green I as described [20] (link). Ten μl reaction volumes in 384-well plates sealed with LightCycler 480 sealing foil (Roche Diagnostics) were processed in LightCycler 480 (Roche Diagnostics) under the following cycling conditions: initial 3 minutes denaturation at 95°C, followed by 50 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. Melting curve analysis was carried out from 72°C to 97°C with 0.2°C increments; Ct values for each sample were determined by automated threshold analysis. Primer pairs used for cDNA amplification are listed in Table 1 . Data were normalized to a housekeeping GAPDH mRNA. The qPCRs for each of the biological triplicates was performed in quadruplicates.
4
RNA Extraction and RT-qPCR Analysis of Splenocytes
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RNA isolation from the splenocytes was performed using the RNeasy minikit procedure (Qiagen).15 (link) Forward and reverse primers were used to amplify the transcripts. Samples (1 μg) of RNA from different experimental groups of mice were first utilized for cDNA synthesis with reverse primers (IDT; Sigma) using Mouse Moloney Leukemia Virus reverse transcriptase (Invitrogen) at 37 °C for 90 min. A common master mixture containing deoxynucleoside triphosphates (Promega) and Taq polymerase (Invitrogen), as well as gene specific primers and 0.25 volume of cDNA, was used for amplification with an Applied Biosystems thermocycler. The cycling conditions for genes of interest were 5 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 40 s, and extension at 72 °C for 40 s. The identities of the PCR amplified gene products were verified by agarose gel electrophoresis. Identical aliquots were processed in parallel without reverse transcriptase to rule out the presence of residual genomic DNA contamination in PCR amplification preparations. Densitometric analyses were done using the Quantity One software (Bio-Rad), ethidium bromide staining, and visualization under a UV transilluminator. For densitometric calculations, the same band area was used to determine band intensity and normalized for HGPRT.
5
Quantification of Lung Inflammation Mediators
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Total RNA in lysed lung tissue was extracted using Trizol Reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA) and reversely transcribed to cDNA by applying mouse Moloney leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). mRNA levels in lung tissue of MAVS, IRF3, NF-κB, IL-6, IL-1β, CYP11B, CYP17A, and CYP21A and of the internal control 18S gene were measured by the Veriti PCR System (Applied Biosystems). The PCR products were fractionated on a 1% agarose gel and visualized by ethidium bromide staining. The band intensity of ethidium bromide fluorescence was measured using an image analysis system (Bio-Rad, Hercules, CA), then quantified with Quantity One analysis software (Bio-Rad, Hercules, CA), and expressed as the ratio to 18S. The sequences of the primers were as follows: 18S, 5′-AGGGGAGAGCGGGTAAGAGA-3′ and 5′-GGACAGGACTAGGCGGAACA-3′; MAVS, 5′-CAGATTGGTCCCAGTAA-3′ and 5′-GCAAGGTCCACAGAGC-3′; IRF3, 5′-AGAGGCTTGTGATGGT-3′ and 5′-GGCTGTTGGAGATGTG-3′; NF-κB, 5′-TTTATCTCGCTTTCGG-3′ and 5′-GCTCCAGTCTGTCCCTC-3′; IL-1β, 5′-GCTGGAGAGTGTGGAT-3′ and 5′-CTTGTGAGGTGCTGATG-3′; IL-6, 5′-CCAACAGACCTGTCTATACCAC-3′ and 5′-GTGACTCCAGCTTATC-3′; CYP11B, 5′-CAGAACTAATGTGTATGT-3′ and 5′-TTGACCAGAGAAGATG-3′; CYP17A, 5′-CTGATACAAGCCAAGAT-3′ and 5′-CTGAAGCCTACATACTG-3′; CYP21A, 5′-CACTTCCTACAGCCTAA-3′ and 5′-CCTCCTCAATGGTTCT-3′.
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