TRIzol reagent (Invitrogen; Thermo Fisher Scientific) was used to extract RNA. Reverse-transcription was performed using a Takara reagent kit (Takara Biotechnology, Otsu, Japan) to obtain cDNA, followed by qPCR amplification using SYBR Green Master Mix (Yeasen, Shanghai, China) according to the manufacturer's protocol. The primers used in this work are as follows: TMEM100, forward primer sequence: 5′-GGTTCTTGTGGAGCCATAGG - 3′ and reverse primer sequence: 5′-CTCTCCTTCTCTTGGTCTCTCT - 3′; GAPDH, forward primer sequence: 5′-GCATCGAGATCCCTCCAAAAT - 3′ and reverse primer sequence: 5′ -GATGTTTGTCATACTTCTCATGG - 3′.
Takara reagent kit
Manufactured by Takara Bio
Sourced in Japan
The Takara reagent kit is a set of laboratory reagents designed for various biochemical and molecular biology applications. The kit contains a collection of essential components necessary for common experimental procedures, such as DNA/RNA extraction, amplification, and analysis. The core function of the Takara reagent kit is to provide researchers with a convenient and reliable solution for their laboratory needs.
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5 protocols using takara reagent kit
1
RNA Extraction and qPCR Analysis
2
Quantitative PCR Analysis of Ghrelin Signaling
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A Takara reagent kit (Takara, Tokyo, Japan) was used to extract the total RNA [17 (link)]. The RNA was reverse-transcribed into cDNA by using a PrimeScript RT reagent kit (Takara). The expression of ghrelin, GHSR1α, GHSR1β, and mTOR RNA was detected by real-time PCR using a quantitative PCR system (ABI 7300, company, city, state, U.S.A.) with SYBR Premix. After the reaction, real-time PCR amplification and thawing curves were confirmed, and the 2−ΔΔCt values were calculated.
3
Kidney RNA Extraction and qPCR Analysis
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Total RNA was extracted from kidney tissues with TRIzol reagent (Invitrogen Life Technologies, CA, USA) which was then reverse transcribed to create cDNA using the Takara reagent kit (Takara Biotechnology, Shiga, Japan), followed by standard methodology SYBR Green master mix (Yeasen, Shanghai, China) qPCR amplification. Relative gene expression was normalized against GAPDH. The primers used were all in Table S1 .
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from tissues and cells using the TRIzol reagent (Invitrogen Life Technologies). Using the Takara reagent kit (Takara Biotechnology), the recovered RNA was then reverse-transcribed into complementary DNA. qRT-PCR analysis was performed with SYBR Green master mix (Yeasen). Additional file 1 : Table S1 displays the qRT-PCR analysis primers used.
5
Quantitative Real-Time PCR for Gene Expression
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Utilizing the TRIzol reagent, total RNA extraction was done (Invitrogen; Thermo Fisher Scientific). Reverse transcription was conducted for cDNA generation with a Takara reagent kit (Takara Biotechnology, Otsu, Japan). qPCR amplification was then done utilizing SYBR Green Master Mix (Yeasen, Shanghai, China) as directed by the manufacturer. In this study, the following primers were utilized: GREM1, forward primer sequence: 5′-GTCACACTCAACTGCCCTGA-3′ and reverse primer sequence: 5′-GGTGAGGTGGGTTTCTGGTA-3′; GAPDH, forward primer sequence: 5′-GTGGACCTGACCTGCGTCT-3′ and reverse primer sequence: 5′-GTGTCGCTGTTGAAGTCAGAGGAG-3′.
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