Berberine (CSN13524) was purchased from CSNpharm (Chicago, IL, USA) and dissolved with dimethyl sulfoxide (DMSO) in 10 mM for storage solution. High-glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA, and human IL-1β recombinant protein (PHC0815) were provided by Gibco (Waltham, MA, USA). Mesenchymal stem cell adipogenic differentiation medium (DM) was obtained from ScienCell (Carlsbad, CA, USA). Phosphate-buffered saline (PBS), Modified Oil Red O Staining Kit (C0158S), DMSO, 4% paraformaldehyde (PFA), and penicillin/streptomycin solution (100×) were acquired from Beyotime (Shanghai, China). TRIzol reagent and RNA-Quick Purification Kit (RN001) were purchased from Invitrogen (Carlsbad, CA, USA) and Shanghai Yishan Biotechnology (Shanghai, China), respectively. Recombinant human TGF-β1 (AF-100-21C) and Fast Western blot kit, ECL Substrate (35050) were provided by PeproTech (Rocky Hill, NJ, USA) and Pierce (Rockford, IL, USA), respectively. The PAGE Gel Fast Preparation Kit was provided by EpiZyme (Shanghai, China). PrimeScript RT Master Mix (RR036A) and TB Green Premix Ex Taq (RR420A) reagent kits were obtained from Takara (Otsu, Japan). Cell Counting Kit 8 (CCK-8) was provided by DOJINDO (Kumamoto, Japan).
Primescript rt master mix rr036a
Manufactured by Takara Bio
Sourced in Japan, China, Switzerland
PrimeScript RT Master Mix (RR036A) is a reagent kit designed for reverse transcription of RNA to cDNA. It contains all the necessary components for the reverse transcription reaction, including the PrimeScript Reverse Transcriptase enzyme, buffer, and dNTPs.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Page gel fast preparation kit, by Ipsen (1 mentions)
Recombinant human tgf β1 af 100 21c, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Step one plus real time pcr rt pcr system, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green kit, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
4 protocols using primescript rt master mix rr036a
1
Berberine Modulation of Adipogenesis
2
Quantitative PCR Analysis of Mouse Liver and Fat
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Total RNA was extracted from the mouse livers and epididymal fat using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA)) and then reverse-transcribed into cDNA using the PrimeScript RT (Master Mix RR036A, TAKARA, Dalian, China) Reagent kit. The gene expression levels were quantified using reverse transcription quantitative PCR (RT-qPCR) (CFX96, Bio-Rad Laboratories, Hercules, CA, USA) with the primers listed in Supplementary Table S3 . The PCR conditions were as follows: 95 °C for 30 s; 40 cycles of amplification (95 °C for 5 s, 60 °C for 30 s); and 72 °C for 30 s. Then, melting curves were acquired stepwise from 55 to 95 °C. Expression levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression levels of target genes were measured using the comparative cycle threshold method.
3
Gene Expression Analysis Protocol
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The cells were washed with PBS, and total RNA was extracted using Quick RNA Micro Prep Kit 11–328M (Zymo Research, Irvine, CA, Unites States). The RNA was analyzed for quality and was quantified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA) and reverse-transcribed to complementary DNA (cDNA) using Takara Prime Script RT Master Mix RR036A (Takara, San Jose, CA, Unites States) in Applied Biophysics 2,720 thermal cycler (Life Technologies, Waltham, MA, Unites States). Gene expression was analyzed using Power SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, Unites States) using qPCR. The primers used were listed in Table 1 ; GAPDH or β-actin was used as a housekeeping gene. Reactions were detected using Step One Plus real-time PCR (RT-PCR) system (Applied Biosystems, Foster City, CA, Unites States). The relative expression values of the target genes were normalized to the housekeeping gene, and the fold change was calculated using the relative quantification (2−ΔΔCT) method. Three biological replicates per treatment group were run with three technical replicates.
4
Quantifying LncRNA Expression in GC
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Total RNA was extracted from tissues (3 GC tissues and 3 normal adjacent tissues) using RNAiso Plus (9109; Takara Biotechnology Co., Ltd.). The concentration and purity of the isolated RNA was determined using TECAN infinite M100 PRO Biotek microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland) and reverse transcription (37°C for 15 min and 85°C for 5 sec) was performed according to the PrimeScript RT Master mix RR036A (Takara Biotechnology Co., Ltd.). qPCR was performed using SYBRGreen kit (cat no. 4367659; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The reaction procedures were as follows: 50°C for 3 min, 95°C for 3 min, 95°C for 10 sec, and 60°C for 30 sec, for 40 cycles, the melting process was 60 to 95°C (increments of 0.5°C for 10 sec). According to the results of bioinformatics analysis, the expressions of 7 lncRNAs, including AC016735.2, RP11-243M5.2, RP11-400N13.2, RP11-400N13.3, AP001626.1, LINC01139 and RP11-54H7.4, were detected with the primers presented in Table I . The expression levels were calculated using the 2−ΔΔCq method (21 (link)).
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