Total RNA in cells or tissues was extracted and separated with TRIzon reagent (Cwbiotech, CW0580) from lysates. For each sample, 500 ng of total RNA was converted to cDNA using High Capacity cDNA Reverse Transcription Kit (Transgene, AT341-02). The relative expression levels of mRNAs, miRNAs and lncRNAs were quantified by qRT-PCR with SYBR Green I (Roche, 4913914001). Detection of the qRT-PCR results was performed on CFX96 Real-Time PCR Detection System (Bio-Rad). All of the primers are provided in Supplementary Table 1 . The level of miR-101a-3p was analyzed by qRT-PCR normalizing with U6, while the levels of lncRNA and mRNA |were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
High capacity cdna reverse transcription kit
Manufactured by Transgene
Sourced in China
The High-capacity cDNA reverse transcription kit is a laboratory equipment product designed for the reverse transcription of RNA into complementary DNA (cDNA). The kit provides the necessary reagents and enzymes to efficiently convert RNA samples into cDNA, which can then be used for various downstream applications, such as gene expression analysis, cloning, or PCR amplification. The kit's core function is to facilitate the reverse transcription process, allowing researchers to generate cDNA from RNA samples.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Real time pcr system, by Bio-Rad (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Sybr green 1, by Roche (1 mentions)
Trizon reagent, by CWBIO (1 mentions)
Pcr thermal cycler dice tp600, by Takara Bio (1 mentions)
Easypure viral dna rna kit, by Transgene (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
Transstart green qpcr supermix kit, by Transgene (1 mentions)
Nanodrop 1000 system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Transstart tip green qpcr supermix, by Transgene (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Gelstain, by Transgene (1 mentions)
Alphaimager hp, by Bio-Techne (1 mentions)
Mycycler pcr thermal cycler system, by Bio-Rad (1 mentions)
Sybr green pcr master mix, by Vazyme (1 mentions)
Lightcycler 480 sequence detection system, by Roche (1 mentions)
9 protocols using high capacity cdna reverse transcription kit
1
Quantification of RNA Transcripts by qRT-PCR
2
Viral RNA/DNA Extraction and Analysis
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An EasyPure®Viral DNA/RNA Kit (TransGen Biotech, Beijing, China) was used to extract viral DNA and RNA. A high-capacity cDNA reverse transcription kit (TransGen Biotech, Beijing, China) and a PCR Thermal Cycler Dice (TP600) (TaKaRa, Japan).were used to perform the reverse transcription of RNA samples to single-stranded cDNA. The resulting cDNA samples were then subjected to a conventional PCR and RT-PCR.
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted from the eWATs and iBATs using TRIzol according to the manufacturer’s instructions. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (AH341; TransGen, Illkirch-Graffenstaden, France), following the manufacturer’s protocol. The qPCR reactions were conducted with TransStart Green qPCR Super mix kit (AQ101; Transgen, Illkirch-Graffenstaden, France) on a CFX96 system (Bio-Rad, Richmond, CA, USA). RNA concentrations of each sample were determined using a NanoDrop 1000 system (Thermo Scientific, Waltham, MA, USA). All RNA samples were within a 260:280 ratio >1.8, and a 260:230>1.8. Relative target mRNA abundance was normalized to that of GADPH. Primer sets for quantitative real-time PCR are summarized in Table 1 .
4
qPCR Analysis of RpS3 Expression
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The total RNA was purified using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The cDNA synthesis was performed using a High Capacity cDNA Reverse Transcription kit (EasyScript, TransGen, Beijing, China). Quantitative polymerase chain reaction (qPCR) was carried out by the QuantStudioTM 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the TransStart Tip Green QPCR SuperMix (TransGen). The reaction conditions of the qPCR cycling program were: 94 °C for 15 s, 60 °C for 30 s, 72 °C for 45 s, then repeating the entire cycle 40 times. For qPCR experiments, 3 technical replicates for each biological sample were performed. The whole procedure, from the RNA isolation to the qPCR, was repeated 3 times. The primers used for qPCR are exhibited in Supplementary Table S1 . The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal reference gene to normalize the qPCR data. When quantifying the expression level of RpS3 in fly, the RpS3 transcript levels at various developmental stages were determined relative to that of the 12–24 h embryo or the adult testis in each group.
5
RT-PCR Gene Expression Quantification
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The RNA was extracted using TRIzol Reagent (Invitrogen, Waltham, MA, USA). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit (Transgen Biotech, Beijing, China). Primers used to measure endogenous expression are summarized in Table S1 . RT-PCRs were performed using a Taq polymerase kit (TransGen Biotech, Beijing, China). Amplification products were confirmed in 2% agarose gel containing GelStain (TransGen Biotech, Beijing, China) for visualization with AlphaImager® HP (Alpha Innotech, San Leandro, CA, USA). RT-qPCRs were performed using TransStartTM Top Green qPCR Super Mix (TransGen Biotech, Beijing, China). Gene expression was detected using the BioRad real-time PCR system. The housekeeping gene Gapdh was employed as a control for analyzing relative mRNA levels. The 2-ΔΔCt method was used to analyze gene expression data. Each sample was examined in triplicate.
6
Quantitative RT-PCR Analysis of AQP7 Expression
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Total cellular RNA was extracted using Trizol (Invitrogen). The ratio of the absorbance at 260 nm and 280 nm (A260/A280) was used to assess RNA purity. Reverse transcription was performed with 0.2 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Beijing TransGen Biotech Co. Ltd., China). PCR was performed using a MyCycler™ Thermal Cycler PCR system (Bio-Rad, Hercules, CA, USA) using: AQP7: forward 5′-GAA CAG TGA GAA AAA GAC CG-3′ and reverse 5′-CCA GAC AAT CCA GAG TTC AT-3′; 18S ribosomal RNA (18S rRNA): forward 5′-TTG GTG GAG CGA TTT GTC TG-3′ and reverse 5′-AAT GGG GTT CAA CGG GTT AC-3′). All reactions involved initial denaturation at 95 °C for 5 min followed by 36 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s. PCR products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and analyzed using the Alpha Innotech software (Bio-Rad). A kinetic study was performed using different numbers of cycles in order to determine the optimal number of cycles for mRNA quantification during the exponential phase. We observed that 36 cycles provided the best results. AQP7 expression was normalized to the 18S rRNA expression level, and was quantified using a standard curve. There was no significant difference in baseline 18S rRNA levels between control and apelin-13- or palmitate-treated adipocytes.
7
Quantitative Real-Time PCR for Gene Expression
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For real-time PCR, total RNA was extracted from frozen or RNA later®-preserved tissue samples using a high-capacity cDNA reverse transcription kit (TransGen Biotech, Beijing, China), according to the manufacturer’s instructions. Amplification of GAPDH cDNA was performed as a control for mRNA content. The following primers were used: PRDX5 forward: 5′-TCCTGGCTGATCCCACTGG-3′, and reverse: 5′-CAGGGCCTTCACTATGCCAT-3′; and PDGF-B forward: 5′-CTCGATCCGCTCCTTTGATGA-3′, and reverse:5′-CGTTGGTGCGGTCTATGAG-3′. Equal amounts of each cDNA were analyzed by real-time PCR with specific primers for PRDX5 and PDGF-B and by EXPRESSSYBR® Green quantitative PCR SuperMix (TransGen Biotech) using a real-time PCR system (Bio-Rad, Hercules, CA, USA). Each sample was measured in triplicate.
8
Quantitative Real-Time PCR Protocol
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Total RNA from cultured cells was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (TransGen Biotech, Beijing, China) from 2 μg of total RNA. Real-time PCR was performed with a SYBR Green PCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). GAPDH was used as an internal control. Each sample was measured in triplicate. Changes in expression were calculated using the 2−ΔΔCt method [37 (link)]. The relative expression ratio is presented as fold change. The primer sequences are shown in Table 1
9
Quantitative real-time PCR protocol
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RNA was isolated with Trizol and single-strand cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Trans-Gen Biotech) according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) analysis was performed by using the SYBR Real-Time PCR Premixture (Takara) under the following conditions: 5 min at 95 C followed by 40 cycles at 95 C for 30 s, 55 C for 40 s, and 72 C for 1 min by using a Roche LightCycler 480 sequence detection system.
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