AST was purchased from Zhibaicui Biotechnology Co., Ltd., and its purity was >98.5%. Complete Freund's adjuvant was purchased from Sigma-Aldrich; Merck KGaA. DMEM was purchased from Gibco; Thermo Fisher Scientific, Inc. Anti-Vimentin antibody (cat. no. ab92547) was purchased from Abcam. PI staining solution was purchased from Beijing Solarbio Science & Technology Co. An MTT assay kit was purchased from Shanghai, BestBio. EZ-10 Total RNA Mini-Preps kit reagent was purchased from Sangon Biotech Co., Ltd. A reverse transcription kit was obtained from ABclonal Biotech Co., Ltd. Antibodies against PDK1 (cat. no. ab110025), AKT (cat. no. ab18785) and phosphorylated (p-)AKT (cat. no. ab38449) were purchased from Abcam. Secondary antibodies (anti-mouse or anti-rabbit; cat. nos. ZB-2301 and ZB-2305) were purchased from Beijing ZSGB-BIO. Cy3-labeled goat anti-rabbit antibody (cat. no. A0516) was purchased from Beyotime Institute of Biotechnology. The pcDNA3.1 plasmid was synthesized by Shanghai GenePharma Co., Ltd. Lipofectamine® 2000 Transfection Reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc.
Reverse transcription kit
Manufactured by ABclonal
Sourced in China
The Reverse Transcription Kit is a set of reagents and enzymes designed for the conversion of RNA into complementary DNA (cDNA). The kit provides the necessary components to perform this reverse transcription process, which is a fundamental step in various molecular biology applications, such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Genious 2x sybr green fast qpcr mix, by ABclonal (2 mentions)
Anti vimentin antibody, by Thermo Fisher Scientific (1 mentions)
Pi staining solution, by Solarbio (1 mentions)
Ez 10 total rna mini preps kit reagent, by Sangon (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Quantitative pcr instrument, by Bio-Rad (1 mentions)
Rna extraction kit, by Vazyme (1 mentions)
Sybr green, by ABclonal (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Cfx96 qpcr instrument, by Bio-Rad (1 mentions)
Cytoplasmic and nuclear rna purification kit, by Norgen Biotek (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
6 protocols using reverse transcription kit
1
Investigating AST-induced Cellular Responses
2
Quantification of YAP Target Genes
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RT-qPCR was used to verify the effect of CHRAC1 knockdown on YAP target genes in MDA-MB-231 and Hela cells. In this study, shNC was used as a control group and CHRAC1 shRNA (1# and 2#) was used as experimental group. Total RNA from cells was extracted according to the instructions of Vazyme RNA extraction Kit (Cat#: RC112-01; Vazyme, Beijing, China). RNA concentration and Purity were detected by Nanodrop spectrophotometer and 1µg RNA was applied to reverse transcription to synthesize complementary DNA (cDNA) using Reverse Transcription Kit (Cat#: RK20429, ABclonal) under the following conditions: 37 °C for 2 min, 55 °C for 15 min, 85 °C for 5 min. The reverse-transcribed cDNA was diluted 5-fold and RT-qPCR was conducted with the SYBR Green qPCR Mix reagent (Cat#: RK21203, ABclonal) on a Bio-Rad quantitative PCR instrument under the following conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as reference gene. The method to calculate the relative expression was 2−ΔΔCt. All of the RT-qPCR reactions were performed in duplicates. RT-qPCR primers sequences are available in supplementary materials.
3
Comprehensive RNA Expression Analysis
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Total RNA was isolated from the purified splenic B cells using chloroform/isopropyl alcohol method. cDNA was synthetized using reverse transcription Kit (Abclonal, #RK20429) according to manufacturer’s instructions. Then real-time PCR was performed on qTower 3 (Germamy) with SYBR green (Abclonal, #RK21203) and the following probes: Gapdh, Gsdmd, Il1β, Il18, Caspase 1, Nlrp3, Pkm2, Hk1, Hk3, solute carrier family member 2 (Slc15a2), Midline 1 (Mid1) and Gsdma2. The primer sequences were listed in Table 1 .
4
Endometrial Cancer m6A-related lncRNA Analysis
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We collected 20 EC tissue and 11 normal endometrial tissue specimens. These specimens were obtained from surgical patients at Wuhan Union Hospital and were performed in strict accordance with the relevant regulations of medical ethics (Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology), and informed consent was obtained from the participating patients.
To further verify the expression levels of m6A-related lncRNAs in the tissue samples, we extracted total RNA using TRIzol (RNAiso Plus, Takara, Japan) and cDNA was obtained using a reverse transcription kit (ABclonal, Wuhan, China). Expression of three m6A-related lncRNAs was calculated using 2-CT values, and GAPDH mRNA expression was used as a reference. The primer sequences used in this study were as follows: lncSCARNA9 forward 5ʹ-AGTCTTTCCAGTCTACCTGATGC-3ʹ and reverse 5ʹ-CATTGCCCAGAAATGATTAGGCT-3ʹ; lncTRAF3IP2 -AS1 forward 5ʹ-ACTGGTTGACAGAGCACCAAC-3ʹ, and reverse 5ʹ-AAATCCCATCCGTCCTTGCCT; lncAL133243.2 forward 5ʹ- AGTCCACCATTGCTCAACCGA-3ʹ; and reverse 5ʹ-ATCGGCCTTACATCTCCTGGC-3ʹ.
To further verify the expression levels of m6A-related lncRNAs in the tissue samples, we extracted total RNA using TRIzol (RNAiso Plus, Takara, Japan) and cDNA was obtained using a reverse transcription kit (ABclonal, Wuhan, China). Expression of three m6A-related lncRNAs was calculated using 2-CT values, and GAPDH mRNA expression was used as a reference. The primer sequences used in this study were as follows: lncSCARNA9 forward 5ʹ-AGTCTTTCCAGTCTACCTGATGC-3ʹ and reverse 5ʹ-CATTGCCCAGAAATGATTAGGCT-3ʹ; lncTRAF3IP2 -AS1 forward 5ʹ-ACTGGTTGACAGAGCACCAAC-3ʹ, and reverse 5ʹ-AAATCCCATCCGTCCTTGCCT; lncAL133243.2 forward 5ʹ- AGTCCACCATTGCTCAACCGA-3ʹ; and reverse 5ʹ-ATCGGCCTTACATCTCCTGGC-3ʹ.
5
Relative Quantification of Gene Expression
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Cell Total RNA isolation kit (FOERGENE, Chengdu, China) was used to extract total RNA, and then reverse-transcribed into cDNA by Reverse transcription kit (ABclonal, Wuhan, China). Genious 2x SYBR Green Fast qPCR mix (ABclonal, Wuhan, China) was used to perform qPCR and detected by Bio-Rad CFX96 qPCR instrument (Bio-Rad, Hercules, CA, USA). In a previous study, we found that PFDN5 and TBP genes were the most stable reference genes for BAT to WAT transformation through RNA-seq [35 (link)]. Thus, TBP and PFDN5 genes were applied as reference genes. Primers used in this study were shown in Table S1 . The relative expression levels of the target genes were normalized relative to the expression of PFDN5 and TBP using the 2−ΔΔCT method. Six biological replicates and three technical replicates in a biological sample were used for qPCR.
6
Quantification of mRNA Expression in Adipocytes
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Total RNA was extracted from adipocytes and tissues by TRIzol (Invitrogen, CA, USA) and 1 µg RNA was amplified to cDNA with the Reverse Transcription kit (ABCLONAL, Wuhan, China). The qPCR was carried out with 5 μL of Genious 2X SYBR Green Fast qPCR Mix (ABCLONAL, Wuhan, China), 0.8 μL of cDNA, 0.4 μL of primers (Table S2 ), and 3.4 μL ddH2O by the Bio-Rad CFX96 instrument (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was determined by the method of ΔCt. Our previous study showed that PFDN5 is one of the most stable internal genes between BAT and WAT [47 (link)]. In this study, PFDN5 was used as an internal reference gene. To determine the subcellular localization of FGF11, nuclear and cytoplasmic RNA was extracted using the cytoplasmic and nuclear RNA purification kits (Norgen Biotek, Thorold, ON, Canada).
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