Except the RNA samples for sequencing, the other RNA was extracted with the RNApure Extraction Kit (Bioteke, Beijing, China) according to the manual of the Kit. The methods of reverse transcription and RT-PCR were performed according to Zhao et al. [98 (link)]. The threshold cycle (CT) value was automatically calculated by the Bio-Rad CFX Manager 3.1 system software, and the delta-delta Ct method was used to calculate the relative expression levels [99 (link), 100 (link)]. The rice eEF-1α gene was used as an internal control for quantitative RT-PCR, while Actin gene for semi-quantitative RT-PCR [54 (link)]. The primers were listed in the Additional file 24 : Table S16.
Rnapure extraction kit
Manufactured by BioTeke
Sourced in China
The RNApure extraction kit is a laboratory tool designed to isolate and purify RNA from a variety of sample types. It utilizes a specialized chemical-based extraction process to effectively separate RNA from other cellular components, allowing for its collection and further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Exicycler 96 real time quantitative thermal block, by Bioneer (3 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Super m mlv reverse transcriptase, by BioTeke (1 mentions)
M mlv reverse transcriptase, by BioTeke (1 mentions)
M mlv reverse transcription, by Takara Bio (1 mentions)
Nano 2000 ultraviolet spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taq hs perfect mix, by Takara Bio (1 mentions)
Sybr green, by BioTeke (1 mentions)
Sybr green master mix, by Solarbio (1 mentions)
5 protocols using rnapure extraction kit
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantifying mRNA Expression in Carotid Tissue
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The mRNA expression in carotid tissue was detected by real-time PCR. Briefly, total RNA was isolated from carotid tissue using the RNApure extraction kit (BioTeke Corporation, Beijing, China) following the manufacturer’s instructions. The RNA was reversely transcribed to first-strand DNA using Super M-MLV reverse transcriptase (BioTeke Corporation, China). The PCR experiment was performed using the SYBR Green method on the ExicyclerTM 96 Real-Time Quantitative Thermal Block (Bioneer Corporation, Korea). The primer sequences are listed in Table 1 . The results were calculated using the 2-ΔΔCt method and β-actin was used as a control.
3
Quantitative Analysis of Notch Pathway Genes
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Total RNA was extracted from tissues and cells using the RNA pure Extraction Kit (BioTeke Corp., Beijing, China), according to the manufacturer's instructions. RNA sample concentration was measured by UV spectrophotometry and RNAs were reverse-transcribed using M-MLV Reverse Transcriptase (BioTeke Corp.). Primers were synthesized by Sangon Biotech Co., Ltd., (Shanghai, China) and the primer sequences were as follows: Notch1, forward 5′-TGGCTCCATCGTCTACCTG-3′ and reverse 5′-GGCTCCACCGTCTCACTCT-3′; Notch4, forward 5′-GGACTAGGAAATCCCGAACC-3′ and reverse 5′-AACCTCCCGAGCATCAGC-3′; Jagged1, forward 5′-GTGCCGCCATAGGTAGAGT-3′ and reverse 5′-CCAGCCAACCACAGAAAC-3′; Delta-like 1 (DLL1), forward 5′-GGGACGATGTTCGGATAA-3′ and reverse 5′-TCGGCACAGGTAGGAGTT-3′; Mastermind-like protein 1 (MAML1), forward 5′-AGCAACAGTTTCAGCGTCAT-3′ and reverse 5′-GCACAGCAGCAGAAGGTC-3′; p300, forward 5′-ATGATGCCTCGGATGACA-3′ and reverse 5′-GACACTGGTGCTTGACTGC-3′; and β-actin, forward 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′ and reverse 5′-GGCCGGACTCATCGTACTCCTGCTT-3′. Amplification was carried out on an Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer Corp., Daejeon, Korea) and the amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. Results were normalized to β-actin and analyzed using the 2−ΔΔCq formula (17 (link)).
4
Gene Expression Analysis of Metabolic Regulators
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The total RNA was extracted with RNApure extraction kit (BioTeke, Beijing, China), and the concentration was measured with NANO 2000 ultraviolet spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA was reversely transcribed into cDNA with M-MLV reverse transcription (TAKARA, Japan) with Oligo(dT) and random primer. The instruments and reagents used in reverse transcription were RNase-free. The cDNA was used for real-time PCR with Taq HS Perfect Mix (TAKARA) and SYBR Green (BioTeke) to detect the mRNA levels of ANGPTL8, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. β-actin served as the internal control. The PCR procedure was set as follow: 94°C for 5 min 20 s, 60°C for 30 s, 72°C for 40 s, and 40 cycles of 72°C for 5 min 30 s, 40°C for 4 min 30 s, melting 60-90°C every 1°C for 1 s, and incubated at 25°C for several minutes. The PCR ran in Exicycler TM96 quantitative thermal block. The data were calculated using 2-ΔΔCt method. The primers were purchased from Genscript (Nanjing, Jiangsu, China), and the information was shown in Table 1
5
Quantitative Assessment of Gene Expression
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Primers used for detection of the mRNA expression levels of ACVR2A, actin α2 (ACTA2), collagen type I α1 chain (COL1A1) and collagen type IV α1 chain (COL4A1) genes are listed in Table I . Total RNA was isolated from liver samples and cells using the RNApure extraction kit (RP1201), after which RNA was reverse transcribed into cDNA using Super M-MLV reverse transcriptase (PR6502) (both from BioTeke Corporation, Beijing, China) according to the manufacturer's protocol. Subsequently, qPCR was performed in a mix containing primer pairs (10 μM, 0.5 μl of each), cDNA (1 μl), SYBR-Green mastermix (10 μl, SY1020; Beijing Solarbio Science & Technology Co., Ltd.) and ddH2O (8 μl) on an Exicycler™ 96 real-time quantitative thermal block (Bioneer Corporation, Daejeon, South Korea). The relative mRNA expression levels of each gene were calculated using the 2−ΔΔCq method (25 (link)). β-actin was used as a control. The thermocycling conditions were as follows: 94°C for 5 min, 38 cycles at 94°C for 15 sec/60°C for 20 sec/72°C for 30 sec, 72°C for 150 sec, 40°C for 90 sec, melting from 60°C to 94°C (every 1°C for 1 sec), 25°C for 60–120 sec.
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