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M mulv

Manufactured by New England Biolabs
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M-MuLV (Moloney Murine Leukemia Virus Reverse Transcriptase) is a DNA polymerase enzyme that catalyzes the synthesis of complementary DNA (cDNA) from an RNA template. It is commonly used in reverse transcription reactions to generate cDNA from RNA samples for downstream applications such as PCR, gene expression analysis, and cloning.

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Lab products found in correlation

Nucleozol reagent, by Takara Bio (3 mentions) Trizol reagent, by Thermo Fisher Scientific (3 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions) Rneasy mini kit, by Qiagen (2 mentions) Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Oligo dt primer, by New England Biolabs (1 mentions) Turbo dna free, by Thermo Fisher Scientific (1 mentions) Rneasy minielute cleanup kit, by Qiagen (1 mentions) Peqgold plant rna kit, by Avantor (1 mentions) Universal pcr master mix reagents kit, by Thermo Fisher Scientific (1 mentions) Taqman detection system, by Thermo Fisher Scientific (1 mentions) 7500 fast machine, by Thermo Fisher Scientific (1 mentions) Dnase treated, by Promega (1 mentions) Sybr green master mix, by Thermo Fisher Scientific (1 mentions) Quantstudio 6 flex, by Thermo Fisher Scientific (1 mentions) Smart cdna library construction kit, by Takara Bio (1 mentions) Rq1 dnase, by Promega (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Imager 600, by Cytiva (1 mentions)

Close spelling variants of this product

Protoscript® M-MuLV II First Strand cDNA Synthesis Kit M-MuLV first-strand cDNA synthesis kit CDNA M‐MuLV Reverse Transcriptase M-MuLV RT ProtoScript M-MuLV Taq RT-PCR Kit M-MuLV cDNA Synthesis Kit M-MuLV enzyme ProtoScript Moloney murine leukemia virus (M-MuLV) first-strand cDNA synthesis kit M-MuLV RT Buffer MuLV RT (M-MuLV Reverse Transcriptase,

19 protocols using m mulv

1

Retinal RNA Isolation and Rbpms Splicing

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The total retinal RNA from healthy human donor eyes that were obtained from the National Disease Research Interchange (Philadelphia, PA, USA) was generously provided by Dr. D. B. Farber (UCLA; Young et al. 2011 (link)). The RNA was DNase treated with Turbo DNA-free (Ambion, Austin, TX, USA) and purified with RNeasy MiniElute Cleanup kit (Qiagen, Valencia, CA, USA). The first-strand cDNA from 5 μgs of the total RNA was reverse transcribed with oligo-dT primer and M-MuLV (NEB, Beverly, MA, USA). Rbpms splicing variants were identified by PCR, with primers specific to Rbpms isoforms and first-strand retinal cDNA used as a template.
Ye L., Gu L., Caprioli J, & Piri N. (2018). RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site. Molecular genetics and genomics : MGG, 293(4), 819-830.
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2

Quantitative Real-Time PCR Protocol

Cited 3 times
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Subconfluent cells were subjected to various treatments and total RNA was isolated at the indicated time points using TRIZOL reagent. The reverse transcription was performed by using random hexamer and M-MuLV (New England Biolabs, Ipswich, MA). The RT products were used, and qPCR primers were designed by using the Primer3 Plus. The TqPCR analysis was carried out as described previously [50 (link)–53 (link)]. All transcripts were normalized by using Gapdh as a reference gene [54 (link)–57 (link)]. The qPCR primers are listed in Additional file 1: Table S1.
Zhang L., Luo W., Liu J., Xu M., Peng Q., Zou W., You J., Shu Y., Zhao P., Wagstaff W., Zhao G., Qin K., Haydon R.C., Luu H.H., Reid R.R., Bi Y., Zhao T., He T.C, & Fu Z. (2022). Modeling lung diseases using reversibly immortalized mouse pulmonary alveolar type 2 cells (imPAC2). Cell & Bioscience, 12, 159.
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3

RNA Extraction and Reverse Transcription

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Total RNA was extracted from leaves using the peqGOLD plant RNA kit (Peqlab). For reverse transcription, 2 μg of RNA were incubated in a total volume of 9 μL for 5 min at 65°C and then placed on ice for 2 min. The reverse transcriptase M-MuLV (New England Biolabs), oligo(dT) primers, buffer and dNTPs were added in a final volume of 20 μL according to the manufacturer's instructions. The reaction was incubated at 37°C for 60 min and stopped at 70°C for 10 min. The cDNA for the housekeeping gene UBC21 (ubiquitin-conjugating enzyme 21) was amplified with the primers UBCF/UBCR and for the HCC2 gene with the primers Sco2E1F/Sco2E5R.
Steinebrunner I., Gey U., Andres M., Garcia L, & Gonzalez D.H. (2014). Divergent functions of the Arabidopsis mitochondrial SCO proteins: HCC1 is essential for COX activity while HCC2 is involved in the UV-B stress response. Frontiers in Plant Science, 5, 87.
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4

Eag1 Expression Analysis by RT-PCR

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Total RNA was extracted from cell cultures with TRI-zol reagent. Five micrograms of total RNA was reverse transcribed using the Moloney Murine Leukemia Virus Reverse transcriptase (M-MuLV) (New England BioLabs Inc., Ipswich, MA, USA). RT-PCR was performed with 1 μL of cDNA using the TaqMan™ detection system (Thermo Fisher Scientific) and the Universal PCR Master Mix reagents kit (Thermo Fisher Scientific). Probes previously developed from TaqMan were used to study Eag1 (ID: Hs00924320_m1) and Gusb (ID: Hs00939627_m1, as a constitutive gene) expression. The PCR reaction protocol was 95°C for 15 seconds and 60°C for 1 minute (40 cycles). Data were analyzed with the 2−ΔΔCt method.
Chávez-López M.D., Zúñiga-García V., Hernández-Gallegos E., Vera E., Chasiquiza-Anchatuña C.A., Viteri-Yánez M., Sanchez-Ramos J., Garrido E, & Camacho J. (2017). The combination astemizole–gefitinib as a potential therapy for human lung cancer. OncoTargets and therapy, 10, 5795-5803.
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5

Quantifying miRNA and mRNA Transcripts in Cells

Cited 3 times
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At the endpoint, the transfected cells were subjected to total RNA isolation by using NucleoZOL Reagent (Takara Bio USA, Mountain View, CA) according to the manufacturer’s introduction. For qPCR analysis of mRNA transcripts, total RNA was used for reverse transcription with the hexamer and M-MuLV (NEB). The cDNA products were diluted as templates for qPCR. The qPCR primers were designed by Primer3 Plus program (48 (link)). For assessing the miR expression levels mediated by the three expression systems, the reverse transcription reactions were carried out by using miR-specific reverse primers that were complementary with the 3’-end six nucleotides of mature miR-5p and/or miR-3p, preceded with a 44-nt artificial stem-loop sequence. SYBR Green-based quantitative real-time PCR analysis was performed by following our previously optimized TqPCR protocol (49 (link)). The qPCR reactions were done in triplicate. All expression values were normalized to the reference gene GAPDH expression by using the 2–ΔΔCt method (50 –53 (link)). The sequences of the qPCR primers are listed in Supplemental Table 1.
Fan J., Feng Y., Zhang R., Zhang W., Shu Y., Zeng Z., Huang S., Zhang L., Huang B., Wu D., Zhang B., Wang X., Lei Y., Ye Z., Zhao L., Cao D., Yang L., Chen X., Liu B., Wagstaff W., He F., Wu X., Zhang J., Moriatis Wolf J., Lee M.J., Haydon R.C., Luu H.H., Huang A., He T.C, & Yan S. (2019). A simplified system for the effective expression and delivery of functional mature microRNAs in mammalian cells. Cancer Gene Therapy, 27(6), 424-437.
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6

Comprehensive RNA Extraction and Quantification

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Genomic DNA was isolated as described (32 ). Total RNA was isolated using Trizol reagent (Life technologies) and DNase treated according to manufacturer directions (Promega), primed with random nonamer (New England Biolabs), and reverse transcribed with M-MuLV (NEB). qPCR and qRT-PCR reactions were performed with SYBR green mastermix (Applied Biosystems) and run on an Applied Biosystems 7500 Fast machine. The primers for qPCR and qRT-PCR reactions are found in Supplemental table 2.
DeMicco A., Naradikian M.S., Sindhava V.J., Yoon J.H., Gorospe M., Wertheim G.B., Cancro M.P, & Bassing C.H. (2015). B cell-intrinsic expression of the HuR RNA-binding protein is required for the T cell-dependent immune response in vivo. Journal of immunology (Baltimore, Md. : 1950), 195(7), 3449-3462.
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7

qPCR Analysis of mRNA and miRNA Expression

Cited 4 times
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At the endpoint, the transfected cells were subjected to total RNA isolation by using NucleoZOL Reagent (Takara Bio USA, Mountain View, CA) according to the manufacturer’s introduction. For qPCR analysis of mRNA transcripts, total RNA was used for reverse transcription with the hexamer and M-MuLV (NEB). The cDNA products were diluted as templates for qPCR. The qPCR primers were designed by Primer3 Plus program [48 (link)]. For assessing the miR expression levels mediated by the three expression systems, the reverse transcription reactions were carried out by using miR-specific reverse primers that were complementary with the 3′-end six nucleotides of mature miR-5p and/or miR-3p, preceded with a 44-nt artificial stem-loop sequence. SYBR green-based quantitative real-time PCR analysis was performed by following our previously optimized TqPCR protocol [49 (link)]. The qPCR reactions were done in triplicate. All expression values were normalized to the reference gene GAPDH expression by using the 2–ΔΔCt method [50 (link)–53 (link)]. The sequences of the qPCR primers are listed in Supplemental Table 1.
Fan J., Feng Y., Zhang R., Zhang W., Shu Y., Zeng Z., Huang S., Zhang L., Huang B., Wu D., Zhang B., Wang X., Lei Y., Ye Z., Zhao L., Cao D., Yang L., Chen X., Liu B., Wagstaff W., He F., Wu X., Zhang J., Moriatis Wolf J., Lee M.J., Haydon R.C., Luu H.H., Huang A., He T.C, & Yan S. (2019). A simplified system for the effective expression and delivery of functional mature microRNAs in mammalian cells. Cancer Gene Therapy, 27(6), 424-437.
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8

qRT-PCR Measurement of Gene Expression

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For qRT-PCR measurements, overnight cultures of cells were back-diluted 1000-fold and grown in fresh MCCG media with the appropriate concentration of IPTG. When the culture reached OD600 = 0.3, cells were collected by mixing 5 mL of culture with 5 mL of ice-cold methanol. After 10 minutes on ice, cells were spun down at 4°C for 10 minutes, decanted of excess liquid and resuspended in 100 μL of a solution containing 10 mg/mL Lysozyme, 10 mM Tris-HCl pH 7, and 1 mM EDTA. RNA was extracted using RNeasy Mini Kits (Qiagen) according to the manufacturers protocols for Gram-positive Bacteria. Random hexamer reverse transcription with Moloney Murine Leukemia Virus (M-MuLV) (M0253; NEB) was performed according to the manufacturer’s specifications. Gene expression changes were normalized to sigA. For a list of primers used see Table S2.
Parker D.J., Lalanne J.B., Kimura S., Johnson G.E., Waldor M.K, & Li G.W. (2020). Growth Optimized Aminoacyl-tRNA Synthetase Levels Prevent Maximal tRNA Charging. Cell systems, 11(2), 121-130.e6.
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9

Comprehensive RNA Isolation from Diverse Murine Tissues

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For total RNA isolation, mouse brain, fat (inguinal region), heart, kidney, liver, lung, muscle, spleen, femur and parietal bone (PB) at various ages were harvested immediately after sacrificing the animals, placed in RNase-free mortars containing NucleoZOL Reagent (Takara Bio USA, Mountain View, CA) and liquid nitrogen, and crashed with RNase-free pestles in a RNase-free biosafety cabinet. Total RNA was subsequently isolated according to the manufacturer's introduction, and subjected to reverse transcription with hexamer and M-MuLV (New England Biolabs, Ipswich, MA). The cDNA products were diluted as templates for qPCR.
Liu W., Deng Z., Zeng Z., Fan J., Feng Y., Wang X., Cao D., Zhang B., Yang L., Liu B., Pakvasa M., Wagstaff W., Wu X., Luo H., Zhang J., Zhang M., He F., Mao Y., Ding H., Zhang Y., Niu C., Haydon R.C., Luu H.H., Wolf J.M., Lee M.J., Huang W., He T.C, & Zou Y. (2019). Highly expressed BMP9/GDF2 in postnatal mouse liver and lungs may account for its pleiotropic effects on stem cell differentiation, angiogenesis, tumor growth and metabolism. Genes & Diseases, 7(2), 235-244.
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10

Gene Expression Analysis by qPCR

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Total RNA was extracted by TRIzol Reagent (Thermo, 15596-018), and reverse transcription was performed using reverse-transcriptase M-MuLV (NEB, M0253L) to generate cDNA, followed by. qPCR analysis using SYRB Green qPCR Mix (Roche, A0001) by QuantStudio6 Flex (Thermo). The expression level of each gene was normalized to that of GAPDH. Primers were listed in Table S2.
Wang H., Huang Y., Yu M., Yu Y., Li S., Wang H., Sun H., Li B., Xu G, & Hu P. (2021). Muscle regeneration controlled by a designated DNA dioxygenase. Cell Death & Disease, 12(6), 535.
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