Mulv reverse transcriptase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, Lithuania, United Kingdom, Spain

MuLV reverse transcriptase is an enzyme that catalyzes the conversion of single-stranded RNA into single-stranded DNA. It is a key component in the process of reverse transcription, which is a fundamental technique in molecular biology and biotechnology.

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Lab products found in correlation

176 protocols using mulv reverse transcriptase

Quantifying lncRNA Expression in HCAECs

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Total RNA from cultured HCAECs was isolated using miRNeasy kits (Qiagen China Co., Ltd.) according to the manufacturer's protocol, followed by purification with TURBO DNA-free System (Ambion; Thermo Fisher Scientific, Inc.). For measuring lncRNAs, RT was performed on 1,000 ng total RNA using MulV reverse transcriptase (Thermo Fisher Scientific, Inc.) and random hexamer primers (Thermo Fisher Scientific, Inc.) in a 20-µl reaction [2 µl RNA (500 ng/µl), 1 µl MulV reverse transcriptase (50 U/µl), 1 µl hexamer primers (100 µM), 2 µl 10X RT reaction buffer, and 14 µl RNAse-free water] and incubated at 42˚C for 1 h. cDNA was used as template for qPCR using an ABI-Prism 7700 Sequence Detection system and the Fast SYBR Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). Human ribosomal P0 (RPLP0) mRNA was as the reference gene. Primer sequences used are as follows: LincRNA-p21, 5'-CCT GTC CCA CTC GCT TTC-3' (forward) and 5'-GGA ACT GGA GAC GGA ATG TC-3' (reverse); LOX-1, 5'-TTA CTC TCC ATG GTG GTG GTG CC-3' (forward) and 5'-AGC TTC TTC TGC TTG TTG CC-3-3' (reverse); RPLP0, 5'-TCG ACA ATG GCA GCA TCT AC-3' (forward) and 5'-ATC CGT CTC CAC AGA CAA GG-3' (reverse). Analysis of relative gene expression levels was performed using the formula 2 -ΔCq , with ΔCq=Cq (target gene) -Cq (control) (19) .

Zhou T, & Chen X. (2017). Long intergenic noncoding RNA p21 mediates oxidized LDL‑induced apoptosis and expression of LOX‑1 in human coronary artery endothelial cells. Molecular medicine reports, 16(6).

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Quantifying Rat Brain mRNA Levels

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Quantification of mRNA was conducted using reverse transcription PCR and primers specifically designed for rat mRNAs Lsd1, Lsd1 + 8a, and Bdnf exon IV (Table 1). RNA was isolated from amygdala tissue as described previously (Kyzar et al., 2017 (link), 2019 (link)) and then reverse transcribed in duplicate using mixed random primers and MuLV reverse transcriptase (Life Technologies). Quantitative real-time PCR was performed using either the Mx3000P qPCR system (Agilent Technologies) and SYBR Green master mix (Fermentas) or a CFX Connect qPCR system with iQ SYBR SuperMix (Bio-Rad). Data were analyzed using the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Hprt1 was used as a reference gene (Kyzar et al., 2017 (link), 2019 (link)). Data are represented as fold change compared to control AIS groups.

Kyzar E.J., Bohnsack J.P., Zhang H, & Pandey S.C. (2019). MicroRNA-137 Drives Epigenetic Reprogramming in the Adult Amygdala and Behavioral Changes after Adolescent Alcohol Exposure. eNeuro, 6(6), ENEURO.0401-19.2019.

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Quantitative Real-Time PCR Protocol

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Total RNA was isolated using the TRIzol extraction method according to manufacturer’s recommendations, then subsequently treated with DNase. One to 2 micrograms of DNase treated-RNA were used to synthesize first strand cDNA after priming with random hexamers (Invitrogen, Waltham, MA, USA) using MuLV reverse transcriptase (Life Technologies, Grand Island, NY, USA) or using a cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time-PCR (qRT-PCR) was carried out as described previously (28 (link)) using SYBR Green master mix (Bio-Rad, Hercules, CA, USA) and transcript-specific primers that have been described previously (25 (link),27 (link),29 (link)–32 (link)). Levels of the mRNA of interest were normalized to the levels of the mRNA for 36B4 (acidic ribosomal phosphoprotein P0) in the same sample. Relative expression was calculated as the difference (ΔCt) between the Ct (threshold cycle) values of the target gene and of 36B4, and expressed as 2^−ΔCt.

Verma S., Prescott R, & Cherayil B.J. (2019). The commensal bacterium Bacteroides fragilis down-regulates ferroportin expression and alters iron homeostasis in macrophages. Journal of leukocyte biology, 106(5), 1079-1088.

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RNA Extraction and qPCR Analysis

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Cells were lysed in 500 μl Trizol, after which total RNA was extracted using the Zymo Research Direct-zol RNA MiniPrep kit with on-column DNase digestion. After extraction, 1 μg of RNA was reverse transcribed using MuLV reverse transcriptase (Life Tech N8080018) in 20 μl reaction, then diluted three-fold with nuclease-free water. 1 μl of cDNA was used in a 15 μl qPCR reaction using KAPA SYBR FAST qPCR Master Mix 2X Universal and quantified on an Eppendorf Realplex2 Mastercycler in triplicate. Relative mRNA expression of genes of interest was quantified using the CT method, normalized to signals from GAPDH. mRNA expression studies were completed in triplicate with error bars representing the standard error of the mean.

Aho E.R., Wang J., Gogliotti R.D., Howard G.C., Phan J., Acharya P., Macdonald J.D., Cheng K., Lorey S.L., Lu B., Wenzel S., Foshage A.M., Alvarado J., Wang F., Shaw J.G., Zhao B., Weissmiller A.M., Thomas L.R., Vakoc C.R., Hall M.D., Hiebert S.W., Liu Q., Stauffer S.R., Fesik S.W, & Tansey W.P. (2019). Displacement of WDR5 from Chromatin by a WIN Site Inhibitor with Picomolar Affinity. Cell reports, 26(11), 2916-2928.e13.

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Gene Expression Analysis by qRT-PCR

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Total RNA was extracted and processed, and qRT-PCR was performed, as previously described21 (link).Total RNA was extracted from the cells using GeneJet RNA Purification Kit (GeneJet RNA Purification Kit: ThermoScientific, K0731). Genomic DNA was removed by incubation with RNase-free DNase I (New England BioLabs, M0303S) in the presence of RNase inhibitor. The RNA was annealed with oligo dt and random hexamer primers, and first strand synthesis was carried out with MuLV reverse transcriptase (Life technologies, Grand Island, NY). Negative controls were performed without reverse transcriptase. A Vii7A (Life technologies, Grand Island, NY) was used to perform the qRT-PCR with ABI TaqMan gene expression assays—Col1A1: Hs00164004_m1, Col3A1: Hs00943809_m1, Col5A1: Hs00609088_m1, and Keratocan: Hs00559942_m1 and the Eukaryotic 18S rRNA Endogenous Control, 4308329. Results were calculated using the ΔΔCt method, using 18S rRNA as the endogenous control.

Karamichos D., Hutcheon A.E., Rich C.B., Trinkaus-Randall V., Asara J.M, & Zieske J.D. (2014). In vitro model suggests oxidative stress involved in keratoconus disease. Scientific Reports, 4, 4608.

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Quantitative Gene Expression Analysis by qPCR

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Selected genes of interest were analyzed for their relative expression changes using quantitative Real-Time PCR (qPCR). First, reverse transcription was performed in 20 μl reaction mixture containing 1 μg of RNA, 1x PCR buffer, 5 mM MgCl2, 10 mM of each dNTP, 0.625 μM oligo dT₁₆, 1.875 μM random hexamers, 20 U RNase inhibitor and 50 U MuLV reverse transcriptase (Life Technologies). The cycles for the reverse transcription were set as follows: 25°C for 10 min, 42°C for 1 h, followed by 99°C for 5 min. The resulting cDNA was amplified in duplicate by qPCR in 10 μl reaction mixture with 200 nM of each specific primer (Table S14) and 1x Fast Syber Green qPCR MasterMix (Life Technologies). The amplification reaction was performed with QuantStudio 7 Flex (Applied Biosystems, Life Technologies). The amplification program was set as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, 60°C for 15 s, 72°C for 20 s. Gapdh and Rplp2 served as housekeeping genes and their amplification data were averaged and used for sample normalization. Excel (Microsoft Office) was used for the comparative quantification analysis.

Gericke C., Mallone A., Engelhardt B., Nitsch R.M, & Ferretti M.T. (2020). Oligomeric Forms of Human Amyloid-Beta(1–42) Inhibit Antigen Presentation. Frontiers in Immunology, 11, 1029.

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Plumericin Inhibits Endothelial Inflammation

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Plumericin was isolated from bark material of Himatanthus sucuuba, as described earlier66 (link). PHA-408 was obtained from Axon Medchem BV (Groningen, Netherlands) and NAC, DPI, Triton X-100, medium M199, fetal bovine serum (FBS), goat serum, gelatin, and paraformaldehyde (PFA) were purchased from Sigma Aldrich (Saint Louis, USA). Penicillin, streptomycin, fungizone, and trypsin-EDTA were bought from LONZA (Visp, Switzerland), endothelial cell growth supplement (ECGS) with heparin from PromoCell (Heidelberg, Germany), and human recombinant TNFα from PeproTech (Vienna, Austria). The products 2′,7′-dichlorodihydrofluresceindiacetate (H2-DCF) and TMB ELISA substrate were obtained from ThermoFischer Scientific (Vienna, Austria). Hoechst 33342 and Alexa 555 were obtained from Life Technologies (Columbus, USA), mouse IgG HRP-linked antibody from GE Health care (Little Chalfont, UK), anti-CD31 and anti-p16 from BioLegend (San Diego, USA), anti-p65 from Santa Cruz (Santa Cruz, USA), anti-Ki-67 from ThermoFischer Scientific, anti-p21 from BD Bioscience (Franklin Lakes, USA), anti-E selectin and anti-ICAM-1 from R&D Systems (Abingdon, UK). Trizol, MuLV-reverse transcriptase, RNAse inhibitor and, oligo dT primers were obtained from Life Technologies, and FastStart SYBR Green Master Mix from ThermoFischer Scientific.

Khan S.Y., Awad E.M., Oszwald A., Mayr M., Yin X., Waltenberger B., Stuppner H., Lipovac M., Uhrin P, & Breuss J.M. (2017). Premature senescence of endothelial cells upon chronic exposure to TNFα can be prevented by N-acetyl cysteine and plumericin. Scientific Reports, 7, 39501.

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Quantitative Real-Time RT-PCR Protocol

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Trizol reagent (T9424), chloroform (C2432), and isopropanol (I9516) were purchased from Sigma-Aldrich (St Louis, MO). Taqman PCR core kit (N8080228), MuLV Reverse Transcriptase (N8080018), and Ribonuclease (RNase) Inhibitor (N8080119) were obtained from Life Technologies (Grand Island, NY). RNase free micro tubes and pipette tips were used in RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR).

Gao C., Gao Z., Greenway F.L., Burton J.H., Johnson W.D., Keenan M.J., Enright F.M., Martin R.J., Chu Y, & Zheng J. (2015). Oat consumption reduced intestinal fat deposition and improved health span in Caenorhabditis elegans model. Nutrition research (New York, N.Y.), 35(9), 834-843.

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RNA Extraction and qPCR Analysis

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Transcript Analysis of Maternal Effect Genes

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Overall transcript levels and poly(A) content of ten ME genes were determined by qRT-PCR. Total RNA from three pools of 20 oocytes from each experimental group was reverse transcribed using MuLV reverse transcriptase (Life Technologies). Overall transcript levels were determined using random hexamer-primed (Life Technologies) cDNA. Oligo(dT)₁₆-primed (Life Technologies) cDNA was used as an indicator for poly(A) tail length. RNA from different oocyte pools was used for random hexamer and oligo(dT)₁₆ priming.
Quantitative RT-PCR was performed with the Light Cycler 480 II (Roche) using either the Universal Probe Liberary (UPL; Roche) or Taqman gene expression assays (Life Technologies) to analyze expression of Brg1 (Smarca4), Tet3, Trim28 (Kap1/Tif1β), Zfp57, Dnmt1, Nlrp2, Nlrp5 (Mater), Nlrp14, Oct4 (Pou5f1) and Zar1. Luciferase was used as an external reference. Primers provided by Biomers, Taqman assays and UPL probes are listed in Table 1. After incubation for 10 min at 95°C, transcripts were amplified using 45 cycles of 10 s at 95°C, 30 s at 60°C and 1 s at 72°C. Each sample was measured in triplicate. The normalized fold change of aged oocytes compared to controls was determined using the 2^-ΔΔCt method.

Dankert D., Demond H., Trapphoff T., Heiligentag M., Rademacher K., Eichenlaub-Ritter U., Horsthemke B, & Grümmer R. (2014). Pre- and Postovulatory Aging of Murine Oocytes Affect the Transcript Level and Poly(A) Tail Length of Maternal Effect Genes. PLoS ONE, 9(10), e108907.

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