M mlv first strand cdna synthesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The M-MLV First-Strand cDNA Synthesis Kit is a reagent system for the reverse transcription of RNA to complementary DNA (cDNA). It utilizes the Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase enzyme to generate first-strand cDNA from total RNA or poly(A)+ RNA templates.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (6 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions) Cfx manager version 3, by Bio-Rad (4 mentions) Plant rna kit, by Omega Bio-Tek (4 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Rneasy micro purification kit, by Qiagen (2 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) P akt, by Abcam (1 mentions) Boster kit, by Boster Bio (1 mentions) Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Boster Bio (1 mentions) Cyp2c9, by Abcam (1 mentions) Cox 1, by Santa Cruz Biotechnology (1 mentions) Cox 2, by Santa Cruz Biotechnology (1 mentions) Erk1 2, by Santa Cruz Biotechnology (1 mentions) β actin, by Boster Bio (1 mentions) Cfx manager, by Bio-Rad (1 mentions) E z n a total rna kit 1, by Omega Bio-Tek (1 mentions) Sybr premix ex taq, by Roche (1 mentions) Ethidium bromide, by Beyotime (1 mentions) Power sybr green master mixture, by Thermo Fisher Scientific (1 mentions)

18 protocols using m mlv first strand cdna synthesis kit

Western Blot and qRT-PCR Analysis of Tumor Proteins

Cited 2 times

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Proteins within tumour tissues and HUVEC cells were extracted using a Boster Kit (Bosterbio, CA, USA) according to the manufacturer’s instructions. Western blotting was performed as previously described [20 (link)]. The antibody, which was raised against the Cyp2c44’s IGRHQPPSMKDKMKC peptide (GenScript), was generated according to previous studies [13 (link), 16 (link)]. Other antibodies used in this study are as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bosterbio), β-actin (Bosterbio), Cyp2c9 (Abcam, Cambridge, UK), COX1 (Santa Cruz, CA, USA), COX2 (Santa Cruz), Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Santa Cruz), ERK1/2 (Santa Cruz), P-AKT (Abcam), AKT (Abcam), and CD31 (Abcam).
For qRT-PCR, RNA from tumours was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed using the M-MLV First-Strand cDNA Synthesis Kit (Invitrogen) [15 (link)]. The mRNA levels of target genes were quantified by qRT-PCR using Power SYBR Green PCR Master Mix (Invitrogen) with the primers listed in Additional file 1: Table S1. GAPDH served as an internal control and the results were analysed using the 2^-ΔΔCt method.

Wu L., Wang W., Dai M., Li H., Chen C, & Wang D. (2019). PPARα ligand, AVE8134, and cyclooxygenase inhibitor therapy synergistically suppress lung cancer growth and metastasis. BMC Cancer, 19, 1166.

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Gene Expression Analysis Protocol

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Total RNA was extracted using the E.Z.N.A.^® Total RNA kit I (Omega Bio-tek) according to the manufacturer's instructions. The quality of the RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). For lncRNA and AKT2 mRNA, cDNA was synthesized using a M-MLV First-Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). RT-PCR was performed at 65°C (10 min), on ice (2 min), 40°C (30 min) and 70°C (10 min). The amplification program was 50°C (2 min), 95°C (2 min), followed by 40 cycles of 95°C (15 sec) and 60°C (30 sec). For microRNAs (miRNAs or miRs), RT-PCR and RT-qPCR were performed using the microRNA reverse transcription and RT-qPCR kit (GenePharma, Inc.). qPCR was carried on a Bio-Rad CFX Manager (Bio-Rad Laboratories, Inc.). The reverse transcription program was 25°C (30 min), 42°C (30 min) and 85°C (30 min). The amplification program was 95°C (3 min), followed by 40 cycles of 95°C (12 sec) and 62°C (40 sec). A relative amount of transcripts was normalized using the 2^−ΔΔCq method with β-actin for large mRNAs and U6 for miRNAs. The specific primers used are listed in Table I.

Wang S., Zheng B., Zhao H., Li Y., Zhang X, & Wen J. (2021). Downregulation of lncRNA MIR181A2HG by high glucose impairs vascular endothelial cell proliferation and migration through the dysregulation of the miRNAs/AKT2 axis. International Journal of Molecular Medicine, 47(4), 35.

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Real-Time PCR Protocol for Gene Expression

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Real-time PCR analyses were performed as previously described [18 (link), 19 (link)]. Briefly, total RNA of the cells was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed with an M-MLV first-strand cDNA synthesis kit (Invitrogen). Indicated mRNA levels were determined by qPCR using SYBR Premix Ex Taq (Roche, Basel, Switzerland), and human GAPDH was used as an internal control.

Chen C, & Zhang X. (2017). IRE1α-XBP1 pathway promotes melanoma progression by regulating IL-6/STAT3 signaling. Journal of Translational Medicine, 15, 42.

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Quantitative Real-Time PCR Analysis of Gene Expression

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Bone marrow mononuclear cells were obtained from total bone marrow by density gradient centrifugation. Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNA samples (500 ng each) were reverse transcribed into cDNA using an M-MLV first strand cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was performed using the 7500 real-time PCR system and Power SYBR Green Master Mixture (both Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions ewre as follows: 95°C for 5 min followed by 40 cycles at 95°C for 30 sec, 52°C for 30 sec, 72°C for 45 sec, and a final step at 62°C for 10 min. GAPDH was used as the internal control gene. Fold changes in relative gene expression were calculated according to the ΔΔCq method (28 (link)). In addition, the amplified products were analyzed by DNA electrophoresis on a 2% agarose gel and the gels were stained wih ethidium bromide (Beyotime Institute of Biotechnology, Shanghai, China). Primer sequences for specific gene amplification are listed in Table I.

Wang L., Lin N, & Li Y. (2019). The PI3K/AKT signaling pathway regulates ABCG2 expression and confers resistance to chemotherapy in human multiple myeloma. Oncology Reports, 41(3), 1678-1690.

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Quantifying UCHL5 mRNA Expression

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For analysis of relative mRNA level, total RNA extraction from cells or tumors was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse-transcribed into cDNA using the M-MLV First-Strand cDNA Synthesis Kit (Invitrogen) (23 (link)). The mRNA levels of the UCHL5 gene were quantified by the qRT-PCR method with Power SYBR Green PCR Master Mix (Invitrogen). The primers are listed in Table 3. An Applied Biosystems 7500 Fast Dx Real-Time PCR instrument (Thermo Fisher, USA) was used in the study. GAPDH was taken as an internal control and the results were analyzed using the 2^−ΔΔCt method.

Liu D., Song Z., Wang X, & Ouyang L. (2020). Ubiquitin C-Terminal Hydrolase L5 (UCHL5) Accelerates the Growth of Endometrial Cancer via Activating the Wnt/β-Catenin Signaling Pathway. Frontiers in Oncology, 10, 865.

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Quantitative Analysis of PLK1 Expression

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All transfected cells were washed twice with PBS and the total RNA was extracted using the TRIzol reagent (Takara, Dalian, China) according to the manufacturer’s protocol. Then, RNA was subsequently reversely transcribed to complementary DNA (cDNA) using M-MLV First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Afterwards, qPCR analysis was performed using Platinum SYBR Green qPCR SuperMix-UDG kits (Invitrogen, Carlsbad, CA, USA) on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).The relative amount of PLK1 normalized to β-actin was calculated according to the 2^−∆∆Ct method. Each sample was run in triplicate. The primer sequences were as follows: 5′-AGCCTGAGGCCCGATACTACCTAC-3′ (PLK1-forward), 5′-ATTAGGAGTCCCACACAGGGTCTTC-3′ (PLK1-reverse) and 5′-GCACAGAGCCTCGCCTT-3′ (β-actin-forward), 5′-GTTGTCGACGACGAGCG-3′ (β-actin -reverse).

Kou Z., Wang X., Yuan R., Chen H., Zhi Q., Gao L., Wang B., Guo Z., Xue X., Cao W, & Guo L. (2014). A promising gene delivery system developed from PEGylated MoS2 nanosheets for gene therapy. Nanoscale Research Letters, 9(1), 587.

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Quercetin qPCR Protocol

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Quercetin was purchased from Sigma (St. Louis, MO). Platinum SYBR Green qPCR SuperMix-UDG, M-MLV First Strand cDNA synthesis kit, and TRIzol reagent were obtained from Invitrogen (Grand Island, NY). All other chemicals were reagent grade or the highest available grade and were purchased from commercial vendors.

Chen R., Lin J., Hong J., Han D., Zhang A.D., Lan R., Fu L., Wu Z., Lin J., Zhang W., Wang Z., Chen W., Chen C, & Zhang H. (2014). Potential toxicity of quercetin: The repression of mitochondrial copy number via decreased POLG expression and excessive TFAM expression in irradiated murine bone marrow. Toxicology Reports, 1, 450-458.

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miRNA Expression Profiling Protocol

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Total RNAs were extracted using Trizol (Invitrogen) according to the manufacturer's instructions and reverse-transcribed by M-MLV First Strand cDNA Synthesis Kit (Invitrogen). The expression of miRNAs was measured by quantitative real-time PCR according to the manufacturer's protocol (Ribobio Co.) using Power SYBR Green PCR Master Mix (Invitrogen), and U6 small nuclear RNA was used as an internal normalized reference. Each reaction was performed in triplicate, and the results were analysed using 2^−ΔΔCt method.

Chen C., Wang Y., Yang S., Li H., Zhao G., Wang F., Yang L, & Wang D.W. (2015). MiR-320a contributes to atherogenesis by augmenting multiple risk factors and down-regulating SRF. Journal of Cellular and Molecular Medicine, 19(5), 970-985.

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Measuring Sirtuin mRNA Levels

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Total RNA was extracted from 50 oocytes using RNeasy micro purification kit (Qiagen), the first strand cDNA was generated with M-MLV first strand cDNA synthesis kit (Invitrogen), using oligo (dT) primers. A cDNA fragment of Sirt1, Sirt2 and Sirt3 were amplified. GAPDH was selected as a reference gene. Primer sequences are shown in Table I. The SYBR Premix Ex Tag2 kit (Takara) was used in an ABI prism 7500 Sequence Detection System. Relative gene expression was calculated by the 2^ΔΔCt method.

Zhang T., Zhou Y., Li L., Wang H.H., Ma X.S., Qian W.P., Shen W., Schatten H, & Sun Q.Y. (2016). SIRT1, 2, 3 protect mouse oocytes from postovulatory aging. Aging (Albany NY), 8(4), 685-694.

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Quantifying Gene Expression in Mollusk Mantle

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Real-time quantitative PCR (RT-qPCR) was designed to verify the differences in gene expression of selected target gene observed during RNA-Seq analysis. The mantle tissues of A. Pleuronectes (Linneus) for RT-qPCR were collected from 5 individuals. Both sides (red and white) of mantle were separately collected. The cDNA was synthesized using M-MLV First Strand cDNA Synthesis Kit (Invitrogen). Primers designed for each gene were present in S1 Table. The qRT-PCR was performed using DyNAmoColorFlash SYBR Green qPCR Kit (Thermo Scientific, USA) according to the manufacturer’s protocol and done on the 7500 Real-time PCR system (ABI Applied Bisosystems, USA). After amplification, fluorescent data was converted to threshold cycle values (CT). The concentration of the template in the sample was determined by relating the CT value to the standard curve. Target gene transcript levels were normalized against reference gene transcripts levels. GAPDH was used as the reference gene. Statistical analysis Quantitative data was expressed as mean ± S.E.M.

Huang R.L., Zheng Z., Wang Q.H., Zhao X.X., Deng Y.W., Jiao Y, & Du X.D. (2015). Mantle Branch-Specific RNA Sequences of Moon Scallop Amusium pleuronectes to Identify Shell Color-Associated Genes. PLoS ONE, 10(10), e0141390.

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