Two step reverse transcription reaction kit

Manufactured by Takara Bio

Sourced in China

The Two-step reverse transcription reaction kit is a laboratory equipment used for the conversion of RNA to complementary DNA (cDNA). This kit facilitates the reverse transcription process, a crucial step in various molecular biology applications, such as gene expression analysis and cDNA library construction.

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Lab products found in correlation

Sybr premix ex taq kit, by Takara Bio (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) 7500 real time pcr system, by Takara Bio (2 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Reverse transcription kit (2398 citations) Two-step reverse transcription kit (5 citations) Reverse transcription reaction (2 citations) Transcription kits (2 citations)

3 protocols using two step reverse transcription reaction kit

Quantitative Analysis of HSP27 mRNA

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Total RNA was extracted from the cells using the Trizol reagent (Invitrogen, Carlsbad, USA) and exactly following the instruction of manufacturer’s manual under RNase-free condition. After complementary DNA was synthesized with a two-step reverse transcription reaction kit (TaKaRa, Dalian, China), qRT-PCR was performed on an Applied Biosystems 7500 Real-time PCR System using SYBR Premix Ex Taq Kit (TaKaRa) in Axygen 96-well reaction plates following the manufacturer’s protocols. Primers were obtained from Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China) and their sequences were:
HSP27 (NM_001540.3), forward primer 5′-AGT GGT CGC AGT GGT TAG-3′, reverse primer 5′-CAG GGA GGA GGA AAC TTG-3′; GAPDH (NM_002046), forward primer 5′-TGG CAC CCA GCA CAA TGA A-3′, reverse primer 5′-CTA AGT CAT AGT CCG CCT AGA-3′. The level of GAPDH mRNA transcript was used to normalize all reported gene expression levels, and the data were analyzed using the 2^-ΔΔCt method.

Zhu Y., Liu Y., Qian Y., Dai X., Yang L., Chen J., Guo S, & Hisamitsu T. (2014). Research on the efficacy of Celastrus Orbiculatus in suppressing TGF-β1-induced epithelial-mesenchymal transition by inhibiting HSP27 and TNF-α-induced NF-κB/Snail signaling pathway in human gastric adenocarcinoma. BMC Complementary and Alternative Medicine, 14, 433.

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Quantifying HIF-1α mRNA in Breast Cancer Cells

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Total RNA from breast cancer cells was prepared with Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Then, cDNA was synthesized using a two-step reverse transcription reaction kit (Takara, Dalian, People’s Republic of China). The levels of HIF-1α gene mRNA transcripts were analyzed by using specific primers and SYBR Green I reagent and the real-time polymerase chain reaction kit, according to the manufacturer’s instructions, using the Bio-Rad iQ5 Quantitative PCR System (Takara). The specific primers for HIF-1α were forward: 5′-AGCCGAGGAAGAACTATGAAC-3′, reverse: 5′-ATTTGATGGGTGAGGAATGGG-3′ and β-actin were forward: 5′-GAGCTACGAGCTGCCTGACG-3′, reverse: 5′-CCTAGAAGCATTTGCGGTGG-3′. The cycling conditions included a holding step at 95°C for 10 minutes, and 35 cycles at 95°C for 25 seconds, 59°C for 30 seconds, and 69°C for 30 seconds. Quantitative real-time PCR analysis was performed using an ABI 7500 Sequence Detector (ABI, Warrington, UK), and the results were analyzed by the method of 2^−ΔΔct.

Zhou Z., Wang S., Song C, & Hu Z. (2016). Paeoniflorin prevents hypoxia-induced epithelial–mesenchymal transition in human breast cancer cells. OncoTargets and therapy, 9, 2511-2518.

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Quercetin Induced Gene Expression

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Following treatment with quercetin, the total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) under RNase-free conditions and reverse transcription was performed in a 20 µl reaction with 200 ng total RNA using a two-step reverse-transcription reaction kit (Takara Biotechnology Co., Ltd., Dalian, China). The RT-qPCR was performed on an Applied Biosystems 7500 Real-time PCR system using a SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) in Axygen 96-well reaction plates.
The primers used in the present study were obtained from Sangon Biotech Co., Ltd. (Shanghai, China) and their sequences were as follows: CSN6 (NM_486571), forward (F) 5′-AGAGGCCACAATGCTGTTTG-3′ and reverse (R) 5′-CGTGGTCTACACCAATGCGTT-3′; GAPDH (NM_002046), F: 5′-TGGCACCCAGCACAATGAA-3′ and R: 5′-CTAAGTCATAGTCCGCCTAGA-3′. GAPDH was used as a housekeeping gene and internal control. The data was analyzed using the 2^−∆∆Cq method (22 (link)).

Yang L., Liu Y., Wang M., Qian Y., Dong X., Gu H., Wang H., Guo S, & Hisamitsu T. (2016). Quercetin-induced apoptosis of HT-29 colon cancer cells via inhibition of the Akt-CSN6-Myc signaling axis. Molecular Medicine Reports, 14(5), 4559-4566.

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