RNA purification was carried out using the High Pure RNA paraffin kit (Roche), following the manufacturer’s instructions. The RNA obtained was suspended in 30 μL of RNA paraffin kit elution buffer and stored at −80 °C until use. The cDNA was prepared with 100 ng of purified RNA, RNAsin 1 U/μL (Promega, USA), 1× buffer TR (Promega, USA), 10 μg/μL random primers (Promega, USA), 20 U/μL MMLV (Promega, USA), and 2 mM dNTPs in a final volume of 20 μL. MMLV negative controls were included. The reaction mixture was incubated at 37 °C for 1 h and stored at −20 °C. RT-PCR was carried out using the primers of Table 1 . β-actin mRNA levels were used for normalization of RNA expression. The amplification conditions were 94 °C for 5 min, followed by 33 cycles of denaturation at 95 °C for 45 s, annealing at 56 °C for 40 s, and extension at 72 °C for 45 s, with a final extension for 5 min at 72 °C. For semi-quantitative analysis, ImageJ software version 1.52a (National Institutes of Health, Bethesda, MD, USA) was used.
M mlv
Manufactured by Promega
Sourced in United States, China, Germany, Spain
M-MLV (Moloney Murine Leukemia Virus Reverse Transcriptase) is a laboratory enzyme used for the reverse transcription of RNA into cDNA. It catalyzes the synthesis of single-stranded cDNA from an RNA template.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (85 mentions)
Trizol, by Thermo Fisher Scientific (51 mentions)
Tri reagent, by Merck Group (14 mentions)
Dnase, by Thermo Fisher Scientific (13 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (10 mentions)
Real time pcr lightcycler 2, by Roche (8 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Dnase 1, by Thermo Fisher Scientific (8 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions)
Sybr premix ex taq kit, by Takara Bio (7 mentions)
Ex taq qpcr premix, by Roche (6 mentions)
Sybr select, by Thermo Fisher Scientific (6 mentions)
Rotor gene 3000, by Qiagen (6 mentions)
Reverse transcriptase, by Promega (6 mentions)
Random primer, by Promega (6 mentions)
Rq1 rnase free dnase, by Promega (6 mentions)
Faststart universal sybr green master, by Roche (5 mentions)
Rnasin, by Promega (5 mentions)
Dnase, by Promega (5 mentions)
Sybr green master mix, by Thermo Fisher Scientific (5 mentions)
277 protocols using m mlv
1
RNA Purification and RT-PCR Analysis
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientific Corp., Waltham, MA, USA). Reverse transcription-PCR was performed with M-MLV (Promega, Madison, WI, USA) following a standard protocol [65°C for 5 minutes (oligonucleotide, random hexamers, 10 mM dnTPs)], followed by 25°C for 10 minutes, 42 °C for 60 minutes and 70°C for 10 minutes (RNase inhibitor, M-MLV, 5X buffer and H2O). The cDNA (1 μL) from each sample was used with SYBR Green in a thermo-cycler (Bio-Rad, Hercules, CA, USA) to perform the real-time PCR in a total volume of 20 μL. Thermal cycling condition was as follows: 1 cycle at 94°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 58°C (depending on the gene) for 30 seconds, and 72°C for 25 seconds. The primers used were as follows: TSGA10 5'-CAACGGCACATGCTATTCTCC-3' (forward), 5'-CCACAGTGCTTATGGTTTCCTTC-3' (reverse); GAPDH 5'-AAGCTCATTTCCTGGTATGACAACG-3' (forward), 5'-TCTTCCTCTTGTGCTCTTGCTGG-3' (reverse). The relative fold change in mRNA expression was calculated using the 2-RΔΔCt method. Average of ΔCt values was normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control. The reactions were performed in triplicates.
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Total RNA was extracted using Trizol reagent (Invitrogen, Thermo Fisher Scientific Corp., Waltham, MA, USA). Reverse transcription-PCR was performed with M-MLV (Promega, Madison, WI, USA) following a standard protocol [65°C for 5 minutes (oligonucleotide, random hexamers, 10 mM dnTPs)], followed by 25°C for 10 minutes, 42 °C for 60 minutes and 70°C for 10 minutes (RNase inhibitor, M-MLV, 5X buffer and H2O). The cDNA (1 μL) from each sample was used with SYBR Green in a thermo-cycler (Bio-Rad, Hercules, CA, USA) to perform the real-time PCR in a total volume of 20 μL. Thermal cycling condition was as follows: 1 cycle at 94°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 58°C (depending on the gene) for 30 seconds, and 72°C for 25 seconds. The primers used were as follows: TSGA10 5'-CAACGGCACATGCTATTCTCC-3' (forward), 5'-CCACAGTGCTTATGGTTTCCTTC-3' (reverse); GAPDH 5'-AAGCTCATTTCCTGGTATGACAACG-3' (forward), 5'-TCTTCCTCTTGTGCTCTTGCTGG-3' (reverse). The relative fold change in mRNA expression was calculated using the 2-RΔΔCt method. Average of ΔCt values was normalized with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control. The reactions were performed in triplicates.
3
Quantifying ITGB6 Expression in Pancreatic Cancer
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A total of six pancreatic cancer cell lines (BXPC-3, CFPAC-1, MIA PaCa-2, ASPC-1, PANC-1 and SW1990) were provided by Shanghai GeneChem Co., Ltd. and cultured in RPMI-1640 basic medium (Corning, Inc.). All cells were routinely subcultured at 37˚C in the presence of 5% CO2 in an incubator with saturated humidity. Total RNA was extracted from the six cell lines using TRIzol reagent according to the manufacturer's instructions. RNA was reverse transcribed to complementary DNA using Promega M-MLV at 42˚C (Promega Corp.). The mRNA expression levels of the ITGB6 gene in different cell lines of interest were detected by quantitative PCR using a LightCycler 480 II (Roche Molecular Systems, Inc.). The composition of the reaction mixture was SYBR premix ex taq 6.0 µl, primer mix 0.3 µl, reverse transcription product 0.6 µl and RNase-free H2O 5.1 µl. The reaction conditions were as follows pre-denaturation at 95˚C for 30 sec, followed by denaturation for 5 sec at 95˚C and annealing for 30 sec at 60˚Cfor a total of 40 cycles. The primer sequences were as follows: ITGB6 forward, 5'-TGATCTTCGCTGTAACCC-3' and reverse, 5'-CAGACCGCAGTTCTTCATA-3'; GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. The experimental results were analyzed by the 2-∆∆Cq method (23 (link)) for relative quantitative analysis.
4
Evaluating Osteogenic Potential of Materials
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Protein expression in the medium was detected using enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Lengton Biotech, China). Transforming growth factor (TGF-β1) and alkaline phosphatase (ALP) were measured according to the manufacturer's instructions. Changes in the ALP, TGF-βRI, and TGF-βRII transcriptional levels on different materials were examined with RT-qPCR. Table 1 lists the primers used for ALP, TGF-βRI, and TGF-βRII. The cDNA species were synthesized using Promega M-MLV (Promega, WI, USA) in accordance with the manufacturer's instructions. Real-time PCR was performed using SYBR® Premix Ex Taq™ (Takara Biotechnology, Shanghai, China) in accordance with the manufacturer's instructions. Upregulated genes were confirmed using a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). Gene expression levels were normalized to GAPDH expression. The relative expression level was calculated by ΔΔCT of the 2–ΔΔCT method [16 (link)].
5
Quantitative gene expression analysis
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RNA was purified by using TRI Reagent (Sigma-Aldrich) and 3 μg was retro-transcripted using MMLV (Promega, Madison, WI, USA). qPCR was performed in triplicate using validated qPCR primers (BLAST), Applied Biosystems™ Power™ SYBR™ Green Master Mix and the QuantStudio3 Real-Time PCR System (Thermo Fisher Scientific, Whaltam, MA, USA). mRNA levels were normalized to RPL8 mRNA and quantified through the 2−ΔΔCt method. The list of primers is reported in Table 1 .
6
Cloning and Characterization of a Novel Morus Gene
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The isolated RNA was used to synthesize cDNA using the reverse transcriptase, M-MLV (Promega). The specific primers (Supplementary Table 2 ) used for PCR amplifications were designed based on the nucleotide sequence of the gene available from the M. notabilis genome (https://www.ncbi.nlm.nih.gov/genome/?term=Morus ) database; the PCR products were separated by electrophoresis and the target DNA fragment was recovered and subcloned into the pMD18-T vector (Invitrogen). After transformation into DH5α, the positive clones were identified and selected for further sequencing. The multiple alignments of the deduced amino acid sequences with the sequences from other plants were conducted using the DNAMAN program. SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/ ) was used with the default parameters to predict signal peptides, and TargetP-2.0 Server (http://www.cbs.dtu.dk/services/TargetP/ ) was used to predict the subcellular localization of proteins. Putative conserved domains were detected using the online NCBI program (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). A phylogenetic tree was generated using the MEGA program by the neighbor-joining method, and bootstrapping was run 1,000 times. The three-dimensional (3D) structure of the protein was generated by the SWISS-MODEL pipeline.
7
Transcriptional Profiling of Antifungal Genes in G. citri-aurantii
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The effects of trans-2-hexenal on the transcriptional profiles of FKS1 (the key gene that synthesizes β-1,3-glucan synthase) and genes related to ergosterol synthesis (ERG1, ERG7, and ERG11) in G. citri-aurantii were evaluated, and the sequences were obtained from a previous RNA-Seq of G. citri-aurantii. RNA was extracted from G. citri-aurantii cells exposed to trans-2-hexenal at concentrations of 0 and 1/2 MIC for 0, 30, 60, and 120 min using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Two micrograms of DNA-free RNA were used for the reverse transcription using M-MLV (Promega, Madison, WI, USA) with oligo dT18. The RT-qPCR was performed on a BIO-RAD CFX Connect Thermal Cycler using FastStart Universal SYBR Green Master (Roche, Basel, Switzerland). All primer pairs for the expression assays are listed in Table 1 . The RT-qPCR was programmed as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and a combined annealing and extension step at 60 °C for 1 min. The 2−ΔΔCT method was used to quantify the value of every sample using the actin gene as an internal reference [24 (link)].
8
Quantitative RT-PCR for Epithelial-Mesenchymal Transition Markers
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Total RNA (800 ng) extracted from each sample was reverse transcribed into cDNA using reverse transcriptase (M-MLV; Promega, Madison, WI, USA). α-Catenin gene expression was quantified using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) on the StepOnePlus system (Applied Biosystems). The expression levels of all genes were normalized to that of human β-actin. The sequences of the primers were as follows: α-catenin (F: 5′-CGCACCATTGCAGACCATTG and R: 5′-GCACCACAGCATTCATCAAGT), β-catenin (F: 5′-GAATGAAGGTGTGGCGACATAT and R: 5′-CAAGTCCAAGATCAGCAGTCTC), E-cadherin (F: 5′-GAAGAAGGAGGCGGAGAAGA and R: 5′-ACACGAGCAGAGAATCATAAGG), ZEB1 (F: 5′-TGTAGAGGATCAGAATGACTC and R: 5′-CAGAATGTAATCGCATGTGT), Snail1 (F: 5′-CTGCTACAAGGCCATGTC and R: 5′-GGACTCTTGGTGCTTGTG), Fascin1 (F: 5′-TTGTGACCTCCAAGAAGAAT and R: 5′-CCCACCGTCCAGTATTTG), β-actin (F: 5′-CCTGGCACCCAGCACAAT and R: 5′-GCTGATCCACATCTGCTGGAA), and TLR4 (F: 5′-CGGAGGCCATTATGCTATGT and R: 5′-TCCCTTCCTCCTTTTCCCTA).
9
Quantitative Analysis of GPR94 Expression
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Total RNA was isolated from hepatocytes from each of the five experimental groups at 24, 48, and 72 h posttreatment using Trizol (Invitrogen of Thermo Fisher Scientific) and then reverse-transcribed into cDNA using reverse transcriptase (M-MLV; Promega Inc., Madison, WI, USA), according to the manufacturers' protocol. cDNA samples were then amplified using Real-Time PCR (LightCycler 480 II; Roche, Basel, Switzerland). Conditions for PCR amplification were set to an initial 95°C for 10 min, which was then followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 35 s. The GPR94 primers used were forward 5′-GACCCTCCAGCAGCATAA-3′ and reverse 5′-AGAAGCCGCTCAACAAAT-3′. The β-actin (control) primers used were forward 5′-GGTCGGAGTGAACGGATTT-3′ and reverse 5′-CTCGCTCCTGGAAGATGG-3′. Quantitative PCR was performed in triplicate, and the relative mRNA expression levels were analyzed using the 2−ΔΔCt method.
10
Apoptosis Gene Expression Analysis by qRT-PCR
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The expression levels of apoptosis-related genes were evaluated by qRT-PCR. First, total RNA was extracted from H2O2-treated BmN-SWU1 cells using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Then, cDNA was synthesized in a 25 μL reaction mixture containing 2 μg total RNA, 2 μL Oligo dT, 2 μL dNTP, 1 μL M-MLV, 1 μL RNasin (Promega) and approximately 12 μL diethylpyrocarbonate (DEPC) water (Beyotime). The resulting cDNA was used in qRT-PCR (Step One Plus, Applied Biosystems, Carlsbad, CA, USA) to analyze the expression of apoptosis-related genes using gene specific primers. Each reaction was performed in a total volume of 15 μL using SYBR Green PCR reagents (TaKaRa, Dalian, China) and incubated for 4 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 31 s, 95°C for 15 s, 60°C for 20 s and 95°C for 15 s. The ribosomal protein L3 (rpL3) was used as an internal control. Primers used in qRT-PCR are listed in Table 1 .
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