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Revertra ace α first strand cdna synthesis kit

Manufactured by Toyobo
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Sourced in Japan, China, United States

The ReverTra Ace-α First-Strand cDNA Synthesis Kit is a laboratory product designed for the reverse transcription of RNA to complementary DNA (cDNA). It provides the necessary components for the efficient conversion of RNA into cDNA, which is a crucial step in various molecular biology and genetic research applications.

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Spelling variants of this product

ReverTra Ace (1031 citations) ReverTra Ace kit (152 citations) ReverTra Ace-α (105 citations) CDNA synthesis kit (98 citations) ReverTra Ace cDNA synthesis kit (16 citations) ReverTra Ace First Strand cDNA Synthesis Kit (9 citations) ReverTra Ace-α-cDNA Synthesis Kit (6 citations) ReverTra Ace first strand synthesis kit (2 citations)

65 protocols using revertra ace α first strand cdna synthesis kit

1

RNA Sequencing and qRT-PCR Analysis

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Total RNA was isolated using NucleoZOL Reagent (MN, Gaithersburg, MD, USA) as per manufacturer’s instruction. For RNA sequencing (RNA-seq), sequencing library was prepared using NEBNext RNA prep kit (NEB, Ipswich, MA, USA). Sequencing was performed using HiSeq 2500 (Illumina, San Diego, CA, USA) with rapid run 150 bp PE mode. Differential expression of OR was confirmed using RPKM (reads per kilo base per million). qRT-PCR analysis was performed according to standard procedure using a ReverTra Ace-α First Strand cDNA Synthesis kit (Toyobo, Osaka, Japan).
qRT-PCR was performed using primers specific for OR51E1 (sense: 5′-TACATTGTGCGGACTGAGCA-3′, antisense: 5′-CCAACTAGCGGTCAAAAGCC-3′), OR51E2 (sense: 5′-TGCATCGTGGTCTTCATCGT-3′, antisense: 5′-TCTGGGTAAGACAGGCCTCA-3′), OMP (sense: 5′-TGTGTACCGCCTCAACTTCA-3′, antisense: 5′-GTCGGCCTCATTCCAATCTA-3′), calcitonin (sense: 5′-CCAGGTGCTCCAACCCC-3′, antisense: 5′-GGCAGCCTCCATGCAGCAC-3′), and GAPDH (sense: 5′-CAAGGTCATCCATGACAACT-3′, antisense: 5′-TTCACCACCTTCTTGATGTC-3′). The cycling conditions were denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s. All reactions were performed in triplicate. Relative mRNA expression was analyzed using the 2−ΔΔCt method, using GAPDH expression for normalization.
Lee H.J., Ku C.R., Cho A., Cho T., Lee C., Kang C.W., Kim D., Cho Y.H., Koo J, & Lee E.J. (2023). Acetate-Mediated Odorant Receptor OR51E2 Activation Results in Calcitonin Secretion in Parafollicular C-Cells: A Novel Diagnostic Target of Human Medullary Thyroid Cancer. Biomedicines, 11(6), 1688.
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2

Quantifying miRNA-1297 Expression

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Total RNA was extracted from cell and tissue samples (hippocampus) using the TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). mRNA (1 ng) was reverse transcribed into cDNA with a ReverTra Ace-α first strand cDNA synthesis kit (Toyobo, Osaka, Japan). The PCR reaction was performed using a qPCR instrument (ABI 7000; Applied Biosystems, Foster City, CA, USA) and the Express SYBR® GreenER™ miRNA qRT-PCR kit (Invitrogen Life Technologies) with the following PCR conditions: 95°C for 10 minutes; 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s; and 72°C for 10 minutes. MiRNA-1297 expression was determined using the 2−ΔΔCt method.
Wang Q., Luo J., Sun R, & Liu J. (2021). MicroRNA-1297 suppressed the Akt/GSK3β signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model. The Journal of International Medical Research, 49(4), 0300060520982104.
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3

Quantification of Gene Expression in Ovary and Uterus

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Ovary and uterus tissues were first harvested before total RNA was extracted using the TRIzol® reagent (Biolab Biotechnology Co., Ltd.). ReverTra Ace-α™ first strand cDNA synthesis kit (Toyobo Life Science) was used for reverse transcription (denaturation at 94°C and annealing at 55°C). qPCR was subsequently performed using the CFX96 Real-Time PCR detection kit (Biolab Biotechnology Co., Ltd.) was used with the following conditions: Initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 20 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. The primer sequences were as follows: p21 forward, 5′-AGTGTGCCGTTGTCTCTTCG-3′ and reverse, 5′-ACACCAGAGTGCAAGACAGC-3′; p53 forward, 5′-AGAGACCGCCGTACAGAAGA-3′ and reverse, 5′-CTGTAGCATGGGCATCCTTT-3′; STK forward, 5′-GTCGCAGGTTCTTGGTCACT-3′ and reverse, 5′-CGAATCTGCACCGTAGTTGA-3′; Bax forward, 5′-AAACTGGTGCTCAAGGCCCT-3′ and reverse, 5′-AGCAGCCGCTCACGGAG-3′; Bcl-2 forward, 5′-AGCGACGAGAGAAGTCATCC-3′ and reverse, 5′-CTGTAGCATGGGCATCCTTT-3′; and GAPDH forward, 5′-GCAAAGTGGAGATTGTTGCC-3′ and reverse, 5′-CCGTATTCATTGTCATACCA-3′. The 2−ΔΔCq method was used to calculate relative expression levels of target genes (33 (link)).
He L., Wang X., Cheng D., Xiong Z, & Liu X. (2020). Ginsenoside Rg1 improves pathological damages by activating the p21-p53-STK pathway in ovary and Bax-Bcl2 in the uterus in premature ovarian insufficiency mouse models. Molecular Medicine Reports, 23(1), 37.
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4

Quantifying HCV and NCL mRNA Expression

Cited 2 times
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Huh7.5.1 or HCVcc-infected Huh7.5.1 cells (2 × 105, respectively) were transfected with 1 μg of different plasmids (pcDNA3.1(–)A, pc-NCL, pSilencer1.0-U6, p-sh-NCL). The total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from the total RNA using the ReverTra Ace-α First strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). RT-qPCR was conducted in optical tubes in a 96-well microtitre plate (Applied Biosystems) with an ABI Step One Plus™ Real-Time PCR system (Applied Biosystems), and the fluorescence signals were generated and recorded during each PCR cycle. The relative mRNA expression of HCV RNA levels at 5′ UTR and NCL mRNA were quantitated using RT-qPCR with the SYBR Green real-time PCR Master Mix kit (Toyobo, Osaka, Japan). The fold change was calculated as 2−dCt, where dCt = Ct (experimental group) – Ct (control group). The primers for HCV RNA at 5′ UTR were HCV-F: 5′-RAYCACTCCCCTGTGAGGAAC-3′ and HCV-R: 5′-TGRTGCACGGTCTACGAGACCTC -3′ (R represents A or G; Y represents C or G) (9 (link),35 (link)). The primers for NCL were NCL-F: 5′-GAAGCCAGCCATCCAA-3′ and NCL-R: 5′-TCTGCCACCAAATCCT-3′. GAPDH ((glyceraldehyde 3-phosphate dehydrogenase), GAPDH-F: 5′-GAAGGTGAAGGTCGGA GTC-3′ and GAPDH-R: 5′GAAGATGGTG ATGGGATTTC-3′) was used as an endogenous control.
Bian W.X., Xie Y., Wang X.N., Xu G.H., Fu B.S., Li S., Long G., Zhou X, & Zhang X.L. (2018). Binding of cellular nucleolin with the viral core RNA G-quadruplex structure suppresses HCV replication. Nucleic Acids Research, 47(1), 56-68.
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5

Quantitative RT-PCR Analysis of Gene Expression

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Total RNAs were isolated from different tissues and three or four samples were pooled together if necessary. One microgram of total RNA was reverse transcribed into cDNA using the ReverTra Ace-α first-strand cDNA Synthesis kit (TOYOBO, Japan). qRT-PCR was performed on a Roche LightCycler 480 real-time PCR system using Realtime SYBR Green I PCR Master Mix (TOYOBO, Japan). Reaction conditions were as follows: denaturation at 95 °C for 1 minute, followed by 40 cycles of 95 °C for 15 seconds, 56 °C for 15 seconds, 72 °C for 20 seconds.
Zheng B., Li S., Liu Y., Li Y., Chen H., Tang H., Liu X., Lin H., Zhang Y, & Cheng C.H. (2017). Spexin Suppress Food Intake in Zebrafish: Evidence from Gene Knockout Study. Scientific Reports, 7, 14643.
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6

qRT-PCR for Gene Expression Analysis

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The total RNA was extracted from the cells in each group according the TRIzol reagent manual (Invitrogen). The total RNA was treated with DNase I (Sigma-Aldrich), quantified and subjected to reverse transcription using the ReverTra Ace-α First Strand cDNA Synthesis Kit (TOYOBO) to generate cDNA. The qRT-PCR was conducted in the RealPlex4 real-time PCR detection system (Eppendorf Co., Germany) using the SyBR Green Real-Time PCR Master Mix (TOYOBO) as the fluorescent dye for nucleic acid amplification. The qRT-PCR included 40 amplification cycles consisting of denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, and extension at 72 °C for 42 s. We used the 2-ΔΔCt calculation method to determine the relative expression of the genes as follows: ΔCt=Ct_genes-Ct_18sRNA and ΔΔCt = ΔCt_all_groups-ΔCt_blankcontrol_group. The mRNA expression levels were normalized according to the expression level of the 18s rRNA gene. The primers used for the amplification of each gene were described in previous studies 8 (link), 9 (link).
Pan Z., Shi Z., Wei H., Sun F., Song J., Huang Y., Liu T, & Mao Y. (2016). Magnetofection Based on Superparamagnetic Iron Oxide Nanoparticles Weakens Glioma Stem Cell Proliferation and Invasion by Mediating High Expression of MicroRNA-374a. Journal of Cancer, 7(11), 1487-1496.
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7

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated from each cell type using the TRIzol Reagent (Invitrogen), according to the manufacturer's protocol. RNA samples were treated with DNase I (Sigma-Aldrich), and quantified and reverse-transcribed into cDNA using the ReverTra Ace-α First Strand cDNA SynthesisKit (TOYOBO). Quantitative real-time PCR (qRTPCR) was conducted using a RealPlex4 real-time PCR detection system (Eppendorf, Germany) with SYBR Green Realtime PCR Master MIX (TOYOBO). qRT-PCR amplification was performed over 40 cycles of denaturation at 95°C for 15 s and annealing at 58°C for 45 s, and the target cDNA was measured using the relative quantification method. A comparative threshold cycle (Ct) was used to determine relative gene expression normalized to the expression of 18S rRNA. For each sample, Ct values were normalized using the formula: ΔCt = Ct_genes - Ct_18S RNA. Relative expression levels were calculated using the formula: ΔΔCt = ΔCt_all_groups - ΔCt_blankcontrol_group. Values used to plot relative gene expression were calculated using the expression, 2-ΔΔCt. Primers used for cDNA amplification are shown in Supplementary Table 2.
Gao S., Wang P., Hua Y., Xi H., Meng Z., Liu T., Chen Z, & Liu L. (2015). ROR functions as a ceRNA to regulate Nanog expression by sponging miR-145 and predicts poor prognosis in pancreatic cancer. Oncotarget, 7(2), 1608-1618.
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8

Gene Expression Analysis by qRT-PCR

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According to previously reported methods (4 (link),12 (link)), the total RNA from cells in all groups were extracted based on the manufacturer instructions for the Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The total RNA was treated with DNase I (Sigma-Aldrich, St. Louis, USA), quantified, and reverse transcribed into cDNA using a ReverTra Ace-α First Strand cDNA Synthesis kit (Toyobo, Shanghai, China; Biotech Co., Ltd., Shanghai, China). Quantitative polymerase chain reaction (qPCR) was performed using a RealPlex4 real-time PCR detection system (Eppendorf Co., Ltd., Hamburg, Germany). A SYBR-Green Real-Time PCR Master Mix (Toyobo) was used as the fluorescence dye for nucleic acid amplification. qRT-PCR was performed for 40 amplification cycles of the following steps: 95°C denaturation for 15 sec, 58°C annealing for 30 sec, and 72°C extension for 42 sec. The relative gene expression levels were calculated and determined using the 2−ΔΔCt method as follows: ΔCt = Ct_genes - Ct_18sRNA and ΔΔCt = ΔCt_all_groups - ΔCt_blank control_group. The mRNA expression levels were calibrated based on the expression level of 18 s rRNA. The primers used are shown in Table I.
Zhao Y., Fei X., Guo J., Zou G., Pan W., Zhang J., Huang Y., Liu T, & Cheng W. (2017). Induction of reprogramming of human amniotic epithelial cells into iPS cells by overexpression of Yap, Oct4, and Sox2 through the activation of the Hippo-Yap pathway. Experimental and Therapeutic Medicine, 14(1), 199-206.
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9

Quantification of CXCR7 Expression

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Total RNA was extracted from the tissues using Trizol reagent (Invitrogen). RNA concentration and purity were measured using a Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies, Houston, TX, USA). cDNA was synthesized from total RNA using the ReverTra Ace-α First-Strand cDNA Synthesis Kit (Toyobo (Shanghai) Biotech Co., Ltd., Shanghai, China). qRT-PCR was performed using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Green Premix Ex Taq (Takara, Dalian, China). β-actin served as internal control. The primer sequences of CXCR7 and β-actin were as follows: CXCR7: 5’-CTATGACACGCACTGCTACATC-3’ (forward), 5’-CTGCACGAGACTGACCACC-3’ (reverse); β-actin 5’-ATGGAGGGGAATACAGCCC-3’ (forward), 5’-TTCTTTGCA GCTCCTTCGTT-3’ (reverse) [14 (link)]. We measured the relative expression of CXCR7 by normalizing with β-actin and using the 2−ΔΔCt method. Each measurement was performed in triplicate.
Yang J., Tang H., Huang J, & An H. (2018). Upregulation of CXCR7 Is Associated with Poor Prognosis of Prostate Cancer. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 5185-5191.
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10

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was isolated with TRIzol reagent (Invitrogen), and complementary DNA (cDNAs) were synthesized by using ReverTra Ace-α-First Strand cDNA Synthesis Kit (Toyobo). qRT-PCR was performed with SYBR Green fluorescence on the StepOnePlus real-time PCR system (Applied Biosystems). The primers were designed using Primer 5 software (Table S2) and synthesized by Sangon Company (Shanghai, China). Gene expression levels were normalized to β-actin mRNA levels and determined by the 2-ΔΔCt method.
Zhou H., Yan L., Huang H., Li X., Xia Q., Zheng L., Shao B., Gao Q., Sun N, & Shi J. (2023). Tat-NTS peptide protects neurons against cerebral ischemia-reperfusion injury via ANXA1 SUMOylation in microglia. Theranostics, 13(15), 5561-5583.
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