HCT-8R cells were treated with IGF-1, AKT inhibitor and 3-MA. Total RNA was extracted using the RNeasy kit (Qiagen GmbH, Hilden, Germany) and transcribed into cDNA with an RNA Reverse Transcriptase kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed with a SYBR®Green PCR assay (Takara Bio, Inc.) according to the manufacturer's protocol (95°C for 1 min, and 40 cycles of 95°C for 5 sec and 60°C for 35 sec, followed by a final standard dissociation protocol), using the primers listed in Table I . Expression of GAPDH served as an internal control. The results were analyzed using the comparative Cq method (2-ΔΔCq) (15 (link)).
Rna reverse transcriptase kit
Manufactured by Takara Bio
Sourced in Japan
The RNA reverse transcriptase kit is a laboratory tool used to convert RNA molecules into complementary DNA (cDNA) strands. This process, known as reverse transcription, is a fundamental step in various molecular biology techniques, such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr assay, by Takara Bio (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Tb green pcr kit, by Takara Bio (1 mentions)
Taqman universal master mix 2 with ung, by Thermo Fisher Scientific (1 mentions)
Abi quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr real time pcr kit, by Takara Bio (1 mentions)
Abi prism 7000, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Total rna kit 2, by Omega Bio-Tek (1 mentions)
5 protocols using rna reverse transcriptase kit
1
Quantitative PCR Analysis of IGF-1 Signaling
2
RNA Extraction and RT-qPCR Quantification
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For RT-PCR analysis, total RNA was isolated with a Total RNA Kit II (OMEGA, BioTek, D6934-01) in RNase-free conditions from cells lysed in RNA lysis buffer and then reverse transcribed into cDNA with RNA reverse transcriptase kit (TaKaRa, RR037A) according to the manufacturer’s protocol. RT-PCR was then performed with a TB Green PCR kit (TaKaRa, RR820A) using the ABI QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems) to determine the expression levels of the genes of interest. The mouse organ tissues were homogenized to detect the virus gene copy number by qPCR (TaqMan™ Universal Master Mix II with UNG, 4440044, Applied Biosystems). All primers were synthesized by the Beijing Genomics Institute (Shenzhen, China). The primer sequences are listed in Supplementary Table 1 .
3
Quantitative Real-time PCR of UCP-2
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The total RNA from abdominal aortic tissue were extracted by Trizol (Life Technologiese Invitrogen, Shanghai, China). A total 1 μg of RNA was used to synthesize cDNA and served as a template for amplification of UCP-2 by RNA reverse transcriptase Kit (Takara, Dalian, China). The forward primer of UCP-2 was 5’-AACAGTCCCAGACAGCCTACA-3’ and the reverse primer was 5’-CCTTCTTTCACTCCCATTTCC-3’ (767bp). The amplification was performed by Quantitative Real-time PCR by a SYBR green premix according to the manufacturer’s guide (SYBR Real-Time PCR Kit, Takara, Dalian, China). Mouse 18S ribosomal RNA were used as endogenous controls. Relative expressions of target genes were standardized to GAPDH, evaluated by the 2-ΔΔCT method and given as a ratio to control the experiments.
4
Quantitative Gene Expression Analysis
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Total RNA was extracted from the TECs using a Total RNA Kit II (Omega Bio-tek, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using an RNA reverse transcriptase kit (TaKaRa, Japan). Real-time quantitative PCR was performed using an ABI PRISM 7000 sequence detection system (Applied Biosystems), and a TaKaRa SYBR Premix Ex Taq II Kit was used for amplification according to the manufacturer’s instructions. The cDNA input was standardized, and 40 cycles of PCR were performed.
5
Quantifying Cytokine Expression in Cells
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The concentration and quality of RNA were determined using an ultraspectrophotometer (model: NANODROP 2000, Thermo Fisher Scientific, USA). The RNA reverse transcriptase kit (Takara; 37°C for 60 min, 85°C for 5 min) was used. The following program was then applied according to the instructions of the expansion kit (Takara, China): 10 s at 95°C, 5 s at 95°C, 20 s at 60°C, 60 s at 95°C, 30 s at 55°C, 30 s at 95°C for 40 cycles. The 2 -ΔΔCT method was used to determine mRNA levels. The primer sequences of IL-6, IL-1β, and MCP-1 are shown in table 1.
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