Extraction of total RNA from cultured cells or serum using Trizol or Trizol LS reagent (Thermo Fisher Scientific, USA) following the guidelines specified by manufacturers, and then quantified using a spectrophotometer K5500 (Thermo Fisher Scientific, USA). For RNA reverse transcription, the reverse transcriptase M-MLV kit (#D2629A, Takara, Japan) was used following the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was done by employing SYBR Green Premix Ex TagTM II kit (#DRR081A, TaKaRa, Japan) on an ABI 7500 Real-Time PCR instrument (Applied Biosystems, USA). The housekeeping gene GAPDH was selected as internal control to normalize the expression level of MIR155HG. The sequences of qRT-PCR primers used in this study were the following: GAPDH: F: 5'-CCTGGTATGACAACGAATTTG-3', R: 5'-CAGTGAGGGTCTCTCTCTTCC-3'; MIR155HG: F: 5'-GGCTCTAATGGTGGCACAAAC-3', R: 5′-ACAGCATACAGCCTACAGCA-3'; U6: F: 5'-GGAACGATACAGAGAAGATTAGC-3', R: 5'-TGGAACGCTTCACGAATTTGCG-3'; ACTIN: F: 5'-GGGAAATCGTGCGTGACATTAAG-3', R: 5'-TGTGTTGGCGTACAGGTCTTTG-3'; NEAT1: F: 5'-AACGCTTTATTTTCCAGGTGGCA-3', R: 5'-CGGGCTTACCAGATGACCAG -3'.
M mlv kit
Manufactured by Takara Bio
Sourced in Japan, China
The M-MLV kit is a reverse transcriptase enzyme system used for the synthesis of first-strand cDNA from RNA templates. The kit includes the M-MLV reverse transcriptase enzyme, reaction buffer, and necessary components for cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Pcr master mix, by Takara Bio (3 mentions)
Sybr green kit, by Roche (2 mentions)
Pmd18 t vector, by Takara Bio (2 mentions)
Sybr green pcr kit, by Roche (2 mentions)
Random 9 mer primers, by Takara Bio (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Sybr green real time pcr assay kit, by Takara Bio (1 mentions)
Cfx96 touch sequence detection system, by Bio-Rad (1 mentions)
Random hexanucleotide primers, by Takara Bio (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Applied biosystems prism 7500, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
22 protocols using m mlv kit
1
RNA Extraction and Quantification Protocol
2
Quantifying Ovarian Maturation Genes
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Nine DEGs were selected from the significantly enriched KEGG pathways to verify the RNA-seq results by qPCR. We also analyzed the expression profiles of these nine genes in five ovarian maturation stages. RNA isolation from tissue and cDNA synthesis were performed using an RNAiso Plus kit and a reverse transcriptase M-MLV kit (TaKaRa) according to the manufacturer’s protocols. The EIF gene of M. nipponense was used as an internal control gene [21 (link)] and the qPCR reaction system and procedures were consistent with our previous study [22 (link)]. Expression levels were calculated by the 2− ΔΔCT method [23 (link)] and the data were analyzed using one-way ANOVA and two-tailed Student’s t-test in SPSS 23.0. Data are presented as the mean ± standard deviation and differences were significant at P < 0.05. The amino acid sequences of two candidate genes were used to generate the phylogenetic trees with MEGA5.0 based on the neighbor joining (NJ) method and bootstrapping replications were 1000.
3
Gene Expression Analysis of Osteogenic Markers
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Total RNA was extracted at various times using TRIzol reagent (Life Technologies), and first-strand cDNA was synthesized using a reverse transcriptase M-MLV Kit (TaKaRa, Shiga, Japan). Gene expression was quantified by RT-qPCR using a SYBR Green kit (Roche, Basel, Switzerland) with gene-specific primers for the detection of PTH1R, runt-related transcription factor 2 (RUNX2), Sp7 (also known as Osterix), and GAPDH. The cycling parameters were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. The primers used in this study were as follows: PTH1R: forward, 5′-AGTGCGAAAAACGGCTCAAG-3′, and reverse, 5′-GATGCCTTATCTTTCCTGGGC-3′; RUNX2: forward, 5′-TGGTTACTGTCATGGCGGGTA-3′, and reverse, 5′-TCTCAGATCGTTGAACCTTGCTA-3′; SP7: forward, 5′-CCTCTGCGGGACTCAACAAC-3′, and reverse, 5′-AGCCCATTAGTGCTTGTAAAGG-3′; and GAPDH: forward, 5′-AGGTCGGAGTCAACGGATTTG-3′, and reverse, 5′-AGGCTGTTGTCATACTTCTCAT-3′. The primer pair for human PTH1R was designed using the Primer Premier 5.0 program based on the cDNA sequence data. The primers for RUNX2, SP7, and GAPDH were previously described by Sakaki-Yumoto et al. [17 (link)], Zhang et al. [18 (link)], and Nozell and Chen [19 (link)].
4
Quantifying RNA Expression in HK-2 Cells
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Total RNA from HK-2 cells was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using a M-MLV kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. Specific primers for α-SMA, collagen I, FSP-1, vimentin, E-cadherin, BMP-7 and GAPDH are listed in Table I . qPCR was performed using a SYBR-Green Real-Time PCR assay kit (Takara Bio, Inc.) on a CFX96 Touch Sequence Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 35 amplification cycles at 95°C for 15 sec and 60°C for 1 min. GAPDH was used as the endogenous control for normalization, and the expression was analyzed using the 2−ΔΔCq method (22 (link)).
5
Quantification of Exosomal lncRNA-GC1 Levels
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The total plasma-derived exosomal RNA was extracted according to the Trizol method. cDNA was synthesized from 30 ng of isolated RNA with a MMLV kit (Takara, Japan). Polymerase Chain Reaction was amplificated with denaturation at 95°C for 5 min, followed by 40 cycles of at 95°C for 10 s, and 60°C for 30 s. The relative levels of circulating exosomal lncRNA-GC1 were normalized using the 2−ΔΔCt method. The sequences of primers used for amplifying lncRNA-GC1 were as follows: sense: F-TGGGGTAACTTAGCAGTTTCAAT-R; antisense: F-GGCAAGCAGTAATCTTACATGACAC-R. The optimal cut-off value of lncRNA-GC1 for prognosis was determined by maximizing the Youden index of the receiver operating characteristic curve (ROC). The stability of exosomal lncRNA-GC1 has been previously identified to remain constant as the total circulating lncRNA-GC1 was packaged within exosomes.24 (link) The time from collection to measurement was within 12 hours after serum collection.
6
SARS-CoV-2 ORF5 Gene Detection
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Total viral RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions and dissolved in RNase-free water. cDNA was synthesized using a M-MLV Kit with random hexanucleotide primers (TaKaRa Co. Dalian, China) following the manufacturer's instructions. A pair of specific primers targeting the ORF5 gene was synthesized based on a previous study (12 (link)) (Table 1 ). Thermocycler conditions used for PCR were 5 min at 95°C followed by 35 cycles of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C, and a final extension of 10 min at 72°C. The products were visualized by electrophoresis on a 1.0% agarose gel containing ethidium bromide. PCR products were purified and cloned into the pMD18-T vector (TAKARA, Japan). Recombinant clones were further sequenced by Tsingke Biotech Co., LTD (Wuhan, China).
7
Transcriptome-Based Prawn RNA Extraction
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Total RNA was extracted from muscle tissue of prawns using TRIzol reagent, according to the manufacturer’s instructions. First strand cDNA synthesis was performed using a reverse transcriptase M-MLV kit (TaKaRa, Japan). Gene-specific primers (Table 1 ) were designed to the 5′- and 3′- ends of cDNA, based on the sequences of partial fragments, using 3′-RACE and 5′-RACE kits following the manufacturer’s instructions (TaKaRa). The partial cDNA sequences were obtained from an RNA-Seq database containing a transcriptome assembly of expressed short reads from M. nipponense (Sun et al., 2015 (link)). PCR products were sequenced on an ABI3730 DNA Analyzer after insertion into the pMD-19T vector.
8
Analyzing OCT4 Target Genes during Erythropoiesis
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Total RNA was isolated by Trizol Reagent (Invitrogen). RNA was then subjected to cDNA synthesis using M/MLV kit (TAKARA) and semiquantitative PCR reactions of OCT4 were performed with PCR master Mix (TAKARA). OCT4 target genes related to erythroid development and pluripotency obtained from ChIP-chip dataset by Boyer et al. [27 (link)] were analyzed by RT-quantitative PCR (RT-qPCR) during erythropoiesis. RT-qPCR was performed using ABI 7300 real-time PCR system (ABI) and samples were normalized to GAPDH with autoset baseline. qPCR was conducted in triplicates using 25 ng of reverse-transcribed cDNA and 0.2 μM of each primer in a 20 μL final reaction volume containing 1 × SYBR Premix EX Taq (TAKARA). PCR cycling conditions were: one cycle of 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 1 min followed by a final cycle of 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. Using comparative critical cycle (Ct) method with GAPDH as an endogenous control, the relative expression was calculated as 2−ΔCt and compared between two groups. qPCR primer sequences were provided in Table 1 .
9
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted using TRIzol (TaKaRa) according to the manufacturer's instructions, and reverse‐transcribed using a reverse transcriptases M‐MLV kit (TaKaRa). qPCR was performed in a real‐time PCR system (Applied Biosystems) using TB Green Premix Ex Taq II (TaKaRa). Cycling conditions were 95°C for 2 minutes, followed by 40 cycles at 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. The primer sequences are shown in Table 1 .
The reactions were conducted three times, and threshold cycle values were normalized to the expression of GAPDH mRNA. The specificity of the products was determined by melting curve analysis. Relative mRNA expression of the target genes was obtained by normalization to the control group and to GAPDH levels with the 2‐△△Ct method.
The reactions were conducted three times, and threshold cycle values were normalized to the expression of GAPDH mRNA. The specificity of the products was determined by melting curve analysis. Relative mRNA expression of the target genes was obtained by normalization to the control group and to GAPDH levels with the 2‐△△Ct method.
10
Quantification of Extracellular Vesicle-Derived lncRNA-GC1
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The total plasma-derived extracellular vesicles-derived RNA was extracted according to the Trizol method. Complementary DNA was synthesized from 30 ng of isolated RNA with an MMLV kit (Takara, Japan). PCR was amplificated with denaturation at 95°C for 5 min, followed by 40 cycles of at 95°C for 10 s, and 60°C for 30 s. The relative levels of circulating extracellular vesicles-derived lncRNA-GC1 were normalized using the 2−ΔΔCt method32 (link). The sequences of primers used for amplifying lncRNA-GC1 were as follows: sense: F-TGGGGTAACTTAGCAGTTTCAAT-R; antisense: F-GGCAAGCAGTAATCTTACATGCAC-R.
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