Revertra ace qrt pcr rt kit

Manufactured by Toyobo

Sourced in Japan

The ReverTra Ace qRT-PCR RT Kit is a laboratory equipment product designed for reverse transcription and real-time PCR. It enables the conversion of RNA to cDNA and the subsequent quantification of gene expression through real-time PCR.

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Lab products found in correlation

Sybr green real time qrt pcr master mix, by Toyobo (2 mentions) Primescript rt reagent kit, by Takara Bio (2 mentions) Nanodrop microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Trizol method, by Thermo Fisher Scientific (1 mentions) Cfx connect real time system, by Bio-Rad (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Sybr green realtime pcr master mix, by Toyobo (1 mentions) Cfx96 real time pcr system, by Bio-Rad (1 mentions) Sepasol, by Nacalai Tesque (1 mentions) Lightcycler 1, by Roche (1 mentions) Taqman universal master mix, by Thermo Fisher Scientific (1 mentions) Sybr green master mix, by Roche (1 mentions) Power sybr green master mix, by CWBIO (1 mentions) Rnapure tissue cell kit, by CWBIO (1 mentions) Ddpcr evagreen supermix, by Bio-Rad (1 mentions) Qx200 droplet generator, by Bio-Rad (1 mentions) Qx200 droplet reader, by Bio-Rad (1 mentions) Rna 6000 nano labchip kit, by Agilent Technologies (1 mentions) Quantasoft software, by Bio-Rad (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

ReverTra Ace qRT-PCR kit ReverTra Ace RT-PCR kit ReverTra Ace qRT Kit ReverTra Ace kit ReverTra Ace QRT-PCR Kit RT-PCR kit

10 protocols using revertra ace qrt pcr rt kit

Quantifying Pathogen-Induced Transcriptional Changes

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An inverted microscope (NIKON ECLIPSE Ti-V) was used to observe the inoculation results 12 h after inoculation. Three A. thaliana roots were cut off and placed in three 1.5 ml Eppendorf tubes at 12 h, 24 h, 36 h, 48 h, 60 h and 72 h after inoculation. The roots were washed using DEPC water and stored at -80°C for later use. Untreated A. thaliana roots were used as a blank control. Each treatment was repeated 3 times.
Microeluted RNA was extracted from each treated A. thaliana root using the MicroElute total RNA kit (OMEGA) according to the manufacturer’s instructions. cDNA was synthesized with the ReverTra Ace qRT-PCR RT Kit (TOYOBO) according to the manufacturer’s instructions. Specific qRT-PCR primers were designed according to Bae et al. (2009) [32 ], and qRT-PCR was performed to assess AOS expression in each of the treatments.

Zhang C., Xie H., Cheng X., Wang D.W., Li Y., Xu C.L, & Huang X. (2015). Molecular Identification and Functional Characterization of the Fatty Acid- and Retinoid-Binding Protein Gene Rs-far-1 in the Burrowing Nematode Radopholus similis (Tylenchida: Pratylenchidae). PLoS ONE, 10(3), e0118414.

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Isolation and Quantification of Kupffer Cell RNA

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The Kupffer cells underwent lysis through the utilization of pre-cooled Trizol on ice for a duration of 5 min, followed by the extraction of their total RNA via chloroform. The RNAs were then precipitated with isopropanol and resuspended in 75% pre-cooled ethanol. The RNA concentration was assessed by means of NanoDrop Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) after dissolution in diethyl pyrocarbonate (DEPC)-treated water. Reverse transcription was performed using the ReverTra ACE qRT-PCR RT Kit (TOYOBO, FSQ-101, Tokyo, Japan), and the resulting cDNA was quantified using Power Green qRT-PCR Mix (Dongsheng Biotech, P2102, Guangzhou, China) under 2^−ΔΔCt method. The primer sequences are given in Table S1. Ultimately, 18S, β-actin and U6 were wholly selected as the internal controls.

Yang Y., Tian T., Li S., Li N., Luo H, & Jiang Y. (2023). LncRNA 220: A Novel Long Non-Coding RNA Regulates Autophagy and Apoptosis in Kupffer Cells via the miR-5101/PI3K/AKT/mTOR Axis in LPS-Induced Endotoxemic Liver Injury in Mice. International Journal of Molecular Sciences, 24(13), 11210.

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Lung Adenocarcinoma miRNA Profile

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We chose miRNA with significant difference and the same trend for further detection. We also detected the miRNA panel in 66 lung benign diseases and 66 lung adenocarcinomas presenting with GGN lesion tissue by qRT-PCR. Total RNA was extracted with an RNA Extraction Kit (SLNco, Cinoasia), and cDNA was synthesized by Prime Script RT reagent Kit (TaKaRa Biotechnology). Primers for the U6 gene and seven miRNAs were obtained from Cinoasia (miR-Quant), validated with PRIMER 5.0 (ABI) and produced by Generay.
The cDNA was reverse transcribed using the ReverTra Ace^® qRT-PCR RT Kit (FSQ-101; Toyobo) kit according to the manufacturer’s protocol (incubation for 15 minutes at 37°C and then 5 seconds at 85°C). qRT-PCR was performed on a Real time Thermo Cycler (FTC3000; Funglyn) with SYBR Green Real-time qRT-PCR Master Mix (QPK-201; Toyobo) as follows: 5 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. The specificity of qRT-PCR product was assessed by melting-curve analysis. Relative Ct values were normalized using the U6 Ct value. Data were analyzed with the 2^−ΔΔCt formula, where ^ΔCt = (C_TmiRNA − C_TU6). Each reaction was performed in triplicate.

He Y., Yang Y., Kuang P., Ren S., Rozeboom L., Rivard C.J., Li X., Zhou C, & Hirsch F.R. (2017). Seven-microRNA panel for lung adenocarcinoma early diagnosis in patients presenting with ground-glass nodules. OncoTargets and therapy, 10, 5915-5926.

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Quantitative RT-PCR Analysis of M. graminicola

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Total RNA was extracted from M. graminicola nematodes at different life stages as described previously^{37 (link)} using the TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA was synthesized using the ReverTra Ace qRT-PCR RT kit (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed with the primer pair MgqRT-PCR-F/MgqRT-PCR-R (Table 1) and actin genes of M. graminicola were amplified as a reference with the primers Mg-ACT-Q-F/Mg-ACT-Q-R^{34 (link)}. The qRT-PCR reactions were performed on a CFX Connect Real-Time System (BIO-RAD, USA) using THUNDERBIRD qRT-PCR Mix (Toyobo, Osaka, Japan). Three technical replicates for each reaction were performed and three independent experiments were performed under the following thermal cycler conditions: 95 °C for 60 sec, 40 cycles at 95 °C for 15 sec, and 60 °C for 30 sec. The relative changes in gene expression were determined using the 2^−ΔΔCT method^{38 (link)}.

Tian Z.L., Wang Z.H., Maria M., Qu N, & Zheng J.W. (2019). Meloidogyne graminicola protein disulfide isomerase may be a nematode effector and is involved in protection against oxidative damage. Scientific Reports, 9, 11949.

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RNA Extraction and RT-qPCR Analysis

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TRIzol reagent (Invitrogen) was used to extract the total RNA from thymuses. Ten micrograms of total RNA from each sample was reverse transcribed using the ReverTra Ace qRT-PCR RT kit (Toyobo, Osaka, Japan). Next, 10 DELs and 10 DEGs were randomly screened for sequencing data verification in all comparison groups. Finally, qRT-PCR was performed using SYBR Green Real-Time PCR Master Mix (Toyobo) on a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, USA). The primers used in the study are listed in Supplementary Table S1.
Actb and
U6 were used as internal controls for mRNA and miRNA to normalize the data, respectively. All experiments were repeated three times, and the relative expression levels of genes were measured according to the cycle threshold (Ct) and calculated using the 2
^–∆∆Ct method.

Li B., Ye Y., Hong L., Li W., Wu Q., Liu W., Ma Y., Xu D, & Li Y. (2023). Transcriptome analysis reveals a potential regulatory mechanism of the lnc-5423.6/IGFBP5 axis in the early stages of mouse thymic involution: lnc-5423.6/IGFBP5 axis regulates thymic involution. Acta Biochimica et Biophysica Sinica, 55(4), 548-560.

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Quantitative Analysis of miRNAs and mRNA Levels

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Total RNA was extracted using the Sepasol (Nacalai Tesque), and level of mature miRNAs was detected using TaqMan MicroRNA systems (Applied Biosystems) using primer specific for each mature miRNA supplied by Applied Biosystems using Light Cycler 1.5 (ROCHE). Briefly, a total of 500 ng RNA were reverse-transcribed with Taqman Reverse-Transcription PCR Kit with specific primer for miR-142-3p. Then, cDNA was mixed with TaqMan Universal Master Mix (Applied Biosystems) and was subjected for real-time PCR. Ct value was analyzed with SDS 2.4 and RQmanager 1.2.1 and quantitated using 2^−ΔΔCt method (Livak, 2001). All data were normalized to endogenous control, the U6 snRNA. Sequences of the primers are T/brachyury 5′-cacaccactgacgcacacggt-3′, 5′-atgaggaggctttgggccgt-3′, Gata4 5′-agccggtgggtgatccgaag-3′, 5′-agaaatcgtgcgggagggcg-3′, Fgf5 5′-gcagtccgagcaaccggaact-3′, and 5′-ggacttctgcgaggctgcga-3′. For quantification of mRNA, total RNA (1 μg) from each sample was used to generate cDNA using ReverTra Ace qRT-PCR RT Kit (Toyobo). Then, cDNA was mixed with Sybr Green Master Mix (ROCHE) and was subjected for real-time PCR using Light Cycler 1.5 (ROCHE). Expression levels of mRNA were compared to known standard samples and normalized to GAPDH.

Abdul Razak S.R., Baba Y., Nakauchi H., Otsu M, & Watanabe S. (2014). DNA Methylation Is Involved in the Expression of miR-142-3p in Fibroblasts and Induced Pluripotent Stem Cells. Stem Cells International, 2014, 101349.

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Quantitative Analysis of miRNA Expression in Lung Adenocarcinoma

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The subset of miRNAs found to be differentially expressed in tumor versus non-tumor analysis by NGS was assayed by quantitative real-time polymerase chain reaction (qRT-PCR) in 73 lung adenocarcinoma patients who presented with GGNs. Total RNA in tumor and adjacent non-tumor tissue was extracted with an RNA Extraction Kit (SLNco, Cinoasia, Shanghai, People’s Republic of China), and cDNA was synthesized by Prime Script RT reagent Kit (TaKaRa Biotechnology, Kusatsu, Japan). Primers for the U6 gene and 23 miRNAs were obtained from Cinoasia (miR-Quant), validated with PRIMER 5.0 (ABI, Foster City, CA, USA) and produced by Generay (Shanghai, People’s Republic of China).
The cDNA was reverse transcribed using the ReverTra Ace^® qRT-PCR RT Kit (FSQ-101; Toyobo, Osaka, Japan) according to the manufacturer’s protocol (incubation for 15 minutes at 37°C and then 5 seconds at 85°C). qRT-PCR was performed on a Real time Thermo Cycler (FTC3000; Funglyn, Ontario, Canada) with SYBR Green Real-time qRT-PCR Master Mix (QPK-201; Toyobo) as follows: 5 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. The specificity of qRT-PCR product was assessed by melting-curve analysis. Relative Ct values were normalized using the U6 Ct value. Data were analyzed with the 2^−ΔΔCt formula, where ^ΔCt = (C_TmiRNA − C_TU6). Each reaction was performed in triplicates.

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Quantitative Analysis of Epithelial-Mesenchymal Regulators

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Total RNA was isolated using an RNA pure Tissue & Cell Kit (CW Biotech, Beijing, China), and cDNA was synthesized using a ReverTra Ace qRT-PCR RT Kit (Toyobo, Osaka, Japan) and miRNA cDNA Synthesis Kit, with Poly(A) (Applied Biological Materials, Richmond, BC, Canada) as per the manufacturer’s protocol. Amplification and detection were performed with Power SYBR Green Master Mix (Cwbiotech) and Gentier 48R System (Tianlong Technology, Xi’an, China). The primers are listed in Supplementary Table S2. For quantitative analysis, the relative mRNA levels of β-catenin, SMAD4, ZEB2, KLF4 and PTEN were normalized to GAPDH, and the relative miRNA level of miR92a was normalized to U6. The primers are listed in Supplementary Table S2.

Li H., Xie C., Lu Y., Chang K., Guan F, & Li X. (2022). Exosomal miR92a Promotes Cytarabine Resistance in Myelodysplastic Syndromes by Activating Wnt/β-catenin Signal Pathway. Biomolecules, 12(10), 1448.

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RNA Extraction and Quantification Protocol

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The yields and purity of extracted RNA were assessed by using a denovix spectrophotometer. The absorption at UV 260 nm was used to assess the RNA yields, and the ratio of 260 nm and 280 nm was used to evaluate RNA purity. An Agilent 4150 Bioanalyzer and the RNA 6000 Nano LabChip kit were employed to calculate the RNA Integrity Number (RIN). The RIN index ranges from 1 to 10, with 1 indicating the greatest degradation and 10 being the most intact RNA (Schroeder et al., 2006 (link)). The copy number of β-actin was detected on a digital droplet PCR (ddPCR) platform. Briefly, RNA in each group was reverse-transcribed into cDNA with the ReverTraAce qRT–PCR RT Kit (TOYOBO, Cat: FSQ-101). The aqueous ddPCR mixture containing 10 µl of ddPCR™ EvaGreen Supermix (BIO-RAD, Cat: #1864033), 3 µl of β-actin primers (3.75 µM) and 7 µl of cDNA was emulsified into picoliter droplets of thermostable oil in a QX200™ Droplet Generator. Subsequently, β-actin was amplified on a QX200 PCR system (Bio-Rad) at 95°C (30 s) and 60°C (60 s) for 40 PCR cycles. The ramp rate between any two consecutive steps was set to 2°C to ensure reliable thermal control. Next, the positive versus negative droplets were read by a QX200™ Droplet Reader, and the absolute quantification of β-actin was calculated using QuantaSoft software (Bio-Rad). Primer sequences for ddPCR are listed in Supplementary Table S5.

He Y., Dong L., Yi H., Zhang L., Shi X., Su L., Gan B., Guo R., Wang Y., Luo Q, & Li X. (2023). Improper preanalytical processes on peripheral blood compromise RNA quality and skew the transcriptional readouts of mRNA and LncRNA. Frontiers in Genetics, 13, 1091685.

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RNA Extraction and qRT-PCR Analysis

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Total RNA from samples was extracted using the RNeasy Mini Kit (QIAGEN). Complementary DNA (cDNA) was obtained using the ReverTra Ace qRT-PCR RT Kit (TOYOBO). Real-time PCR was performed in duplicate using the SYBR Green PCR Master Mix (Roche Diagnostics) on a LightCycler 96 (Roche Diagnostics). Gene expression values for each sample were normalized to the 18 S RNA. qRT-PCR primers are listed in Supplementary Table 5.

Omatsu M., Nakanishi Y., Iwane K., Aoyama N., Duran A., Muta Y., Martinez-Ordoñez A., Han Q., Agatsuma N., Mizukoshi K., Kawai M., Yamakawa G., Namikawa M., Hamada K., Fukunaga Y., Utsumi T., Sono M., Masuda T., Hata A., Araki O., Nagao M., Yoshikawa T., Ogawa S., Hiramatsu Y., Tsuda M., Maruno T., Kogame T., Kasashima H., Kakiuchi N., Nakagawa M.M., Kawada K., Yashiro M., Maeda K., Saito Y., Matozaki T., Fukuda A., Kabashima K., Obama K., Ogawa S., Sheibani N., Diaz-Meco M.T., Moscat J, & Seno H. (2023). THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer. Nature Communications, 14, 5534.

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