Human peripheral blood serum was centrifuged at 3000 RPM (Revolution Per Minute) for 5 minutes and refrigerated at -80°C, total RNA was extracted using TRIpure (RP1001, BioTeke, Beijing) following the manufacturer’s instructions. Mice hepatic tissue and HepG2 cells were lysed, and total RNA was extracted using TRIpure (BioTeke, Beijing). For microRNA analysis, miRNA-specific cDNA was generated with Super M-MLV reverse transcriptase (BioTeke, Beijing). Primer sequences were synthesized in GenScript Biotechnology (Table 1
Tripure
Manufactured by BioTeke
Sourced in China
TRIpure is a complete reagent for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent is a mixture of guanidinium thiocyanate, phenol, and other proprietary components that facilitate the efficient and selective extraction of nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Solarbio (11 mentions)
Super m mlv reverse transcriptase, by BioTeke (9 mentions)
Beyort 2 m mlv reverse transcriptase, by Beyotime (6 mentions)
Taq pcr mastermix, by Solarbio (6 mentions)
Exicycler 96, by Bioneer (4 mentions)
Beyort 2 m mlv, by Beyotime (4 mentions)
Superscript m mlv reverse transcriptase, by BioTeke (2 mentions)
Sybr green, by Merck Group (2 mentions)
Super m mlv reverse transcriptase kit, by BioTeke (2 mentions)
Rnase inhibitor, by BioTeke (2 mentions)
Exicycler 96 pcr instrument, by Bioneer (2 mentions)
Sybr green master mix, by Solarbio (2 mentions)
Exicycler 96 pcr system, by Bioneer (2 mentions)
Sybr green kit, by Solarbio (2 mentions)
Exicycler 96 system, by Bioneer (2 mentions)
Nano 2000, by Thermo Fisher Scientific (2 mentions)
Exicycler 96 rt pcr instrument, by Bioneer (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
33 protocols using tripure
1
Quantitative RNA Expression Analysis
2
RNA Extraction and qRT-PCR Analysis
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TRIpure (RP1001, BioTeke Corporation, China) was used for total RNA or miRNA extraction. Then, total RNA and miRNA were synthesized into cDNA by the SuperScript M-MLV reverse transcriptase (PR6502, BioTeke Corporation) and the miRNA cDNA Synthesis kit (#B532451, Sangon Biotech, Shanghai), respectively. Finally, the qRT-PCR was conducted according to the protocols. GAPDH served as a control. The relative level of the specific mRNAs was analyzed using the 2−ΔΔCT method. The sequences of the primers are listed in Supplementary Table 2.
3
Profiling Circular RNA and miRNA Expression
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Total RNA from all PTC cell lines, clinical tumor, adjacent normal or xenograft tumor tissues were extracted using TRIpure (BioTeke Corporation) and reverse-transcribed into cDNA using a Super M-MLV Reverse Transcriptase kit (BioTeke Corporation) according to the manufacturer's procedure at 42°C for 50 min. RT-qPCR was performed using SYBR Green (Sigma-Aldrich; Merck KGaA). The thermocycling conditions for hsa_circ_0001666 were as follows: 94°C for 5 min, 40 cycles of 94°C for 15 sec, 60°C for 25 sec and 72°C for 30 sec. The thermocycling conditions for miRNAs were as follows: 94°C for 4 min, 40 cycles of 94°C for 15 sec, 60°C for 20 sec and 72°C for 15 sec. The thermocycling conditions for FAM120B and ETV4 were as follows: 94°C for 5 min, 40 cycles of 94°C for 15 sec, 60°C for 25 sec and 72°C for 30 sec. The relative expression levels of hsa_circ_0001666, miR-330-5p, miR-193a-5p, miR-326, FAM120B and ETV4 were determined using the 2−ΔΔCt method (27 (link)). β-actin was used as the internal control for circRNA and mRNA and U6 was used as the internal control for miRNA. The relative gene expression levels in cell lines and xenograft tumors were normalized to the control. All primer sequences are listed in Table SII .
4
Gene Expression Analysis of Heart Tissue
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Total RNA was isolated from heart tissue samples using TRIpure (BioTeke, Wuxi, China) according to the manufacturer’s instructions. Next, RNAs were reverse transcribed to cDNAs using the Super M-MLV reverse transcriptase and RNase inhibitor (BioTeke, Wuxi, China). qRT-PCR was performed using 2×Taq PCR MasterMix and SYBR Green (Solarbio, Beijing, China) in an Exicycler 96 PCR instrument (Bioneer, Daejeon, South Korea). The following primers were utilized to detect the indicated gene transcripts: IFIT3 forward, 5’-ATGGCAGAACTGAGACGAT-3’; IFIT3 reverse, 5’-GTGGTACTCCTGGAGGTTG-3’; IL-1β forward, 5’-CTCAACTGTGAAATGCCACC-3’; IL-1β reverse, 5’-GAGTGATACTGCCTGCCTGA-3’; IL-6 forward, 5‘-ATGGCAATTCTGATTGTATG-3’; IL-6 reverse, 5’-GACTCTGGCTTTGTCTTTCT-3’; TNF-α forward, 5’-CAGGCGGTGCCTATGTCTCA-3’, TNF-α reverse, 5’-GCTCCTCCACTTGGTGGTTT-3’; β-actin forward, 5’-CTGTGCCCATCTACGAGGGCTAT-3’, β-actin reverse, 5’-TTTGATGTCACGCACGATTTCC-3’. The relative gene expression was measured according to the 2-ΔΔCt method. β-actin was considered as the internal control.
5
Quantitative Analysis of Gene Expression
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Total RNA was extracted from HUVECs using TRIpure (BioTeke Corporation, Beijing, China) reagent. cDNA was synthesized with Super M-MLV Reverse transcriptase (BioTeke). Real-time PCR was performed using Power SYBR Green PCR Master Mix (BioTeke) on ExicyclerTM96 fluorometer (Bioneer Corporation, Daejeon, Korea). mRNA levels were calculated using the 2−ΔΔCT method and normalized to the value of β-actin. The primer sequences were presented in Table 1
6
RNA-seq Analysis of Mouse PMVECs
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Total RNA was extracted from the PMVECs of chow and HFD mice using the TRIpure (BioTeke, Beijing, China) and subsequently used for RNA-seq. The data were analyzed using Illumina sequencing platform. The DEGs were determined by |log2 fold change| ≥ 1 and p ≤ 0.05. Furthermore, Reactome, BioCyc and PANTHER, KEGG and GO pathway analyses were conducted to analyze these DEGs.
7
Quantification of RUNX1 Expression
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Total RNA isolation was carried out from infected cells by using the TRIpure (BioTeke, Beijing, China) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed into cDNA with the BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China). Subsequently, amplification was performed by employing the 2 × Taq PCR MasterMix (Solarbio, Shanghai, China) and SYBR Green (Solarbio). The expression of RUNX1 was calculated with 2−ΔΔCt method. β-actin was employed as the internal control. The sequences of primer were as follows. Forward primer: 5′-GACCCTGCCCATCGCTTTC-3′; Reverse primer: 5′-AATCTCGCCACTTGGTTCTTC-3′.
8
RT-qPCR Analysis of Gene Expression
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RNA was isolated from cells using TRIpure (BioTeke Corporation, China) and its concentration was measured by the NanoDrop™ 2000 Spectrophotometer (Thermofisher, USA). RNA was converted to cDNA by performing reverse transcription with the BeyoRT II M-MLV reverse transcriptase (Beyotime, China). Quantitative real-time PCR was performed on the Exicycler™ 96 PCR system (Bioneer, China) using SYBR Green (Solarbio) following the manufacturer’s manual. Primer sequences are available in Table 2 . All data were normalized to ACTB.
Primer sequences for RT-qPCR.
| Sequences (5’-3’) | |
|---|---|
| SH3RF2 Forward | CGTGGTGGTGGAGATGG |
| SH3RF2 Reverse | TGGGAGGTGTAATGTTTGGTG |
| RBPMS Forward | CAAGAACAAACTCGTAGGGACT |
| RBPMS Reverse | GCGGGATAGGTGAAAGC |
| XIAP Forward | CACAGGCGACACTTTCC |
| XIAP Reverse | TTAGCCCTCCTCCACAG |
| MYC Forward | ACACCCTTCTCCCTTCG |
| MYC Reverse | CCGCTCCACATACAGTCC |
9
Quantifying Cardiac Gene Expression
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Total RNA was isolated from heart slice tissues using TRIpure (BioTeke, Beijing, China), and quantified by Nano 2000 spectrophotometry (Thermo, Waltham, MA, USA). The cDNA was prepared with the total RNA using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China). The level of mRNA was measured by qRT-PCR analysis based on SYBR Green kit (Solarbio, Beijing, China) and quantified with the ExicyclerTM 96 apparatus (BIONEER, Daejeon, Korea). The primers were obtained from GenScript Biotechnology Co., Ltd., in Nanjing, China. LAPTM5 primers: forward 5′ GAGTCGCCACCATAGCC 3′ and reverse 5′ TTGAAGGGACAGGAAGG 3′. IL-1β primers: forward 5′ CTCAACTGTGAAATGCCACC 3′ and reverse 5′ GAGTGATACTGCCTGCCTGA 3′. IL-6 primers: forward 5′ ATGGCAATTCTGATTGTATG 3′and reverse 5′ GACTCTGGCTTTGTCTTTCT 3′. TNF-α primers: forward 5′ CAGGCGGTGCCTATGTCTCA 3′ and reverse 5′ GCTCCTCCACTTGGTGGTTT 3′. β-actin in this work was used as an internal standard and the gene expression level was calculated by the 2−ΔΔCt method.
10
Comparative qRT-PCR Analysis of Lung Cell Genes
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Total RNA was extracted from lung tissues and BEAS-2B cells by TRIpure (BioTeke). The RNA was reverse transcribed into cDNA using BeyoRT™ II M-MLV RNase H- (Beyotime). Real-time PCR assay was conducted on Exicycler 96 system (BIONEER) using SYBR Green (Solarbio). The results was calculated using 2-△△CT method. The primer sequences are as follows. Mus musculus ACPL2: F, AATCGCTTCTTGGTGCTG; R, CTACGCTTGGAATGTTGC. Mus musculus RABL4: F, GAAATGCTGGATAAGTTGTG; R, GAGGGAGATGCCTGAAGT. Mus musculus SLC27A3: F, GCATTGTGGGCTGCTTGG; R, GGGCTGGTTGACGAGGTAT. Mus musculus STAU1 F, GTAAAGAAACCAGGAGACG; R, CTGCTGATGGCTAAGATAA.
Mus musculus β-actin: F, CTGTGCCCATCTACGAGGGCTAT; R, TTTGATGTCACGCACGATTTCC. Homo sapiens ACPL2: F, ATGGAGCACTTCAAGGTAA; R, AGCAGAGTAGAGGGCAAA. Homo sapiens RABL4: F, GGAATGGATTTGGTGGTGAA; R, GAGATGCCTGGAGCCTGTGA. Homo sapiens SLC27A3: F, GTTCGGATGGCAAATGAGG; R, TGTACCGGGCAGTTGTGAG. Homo sapiens STAU1 F, ATCCGATTAGCCGACTGG; R, ACTTGAGTGCGGGTTTGG. Homo sapiens β-actin: F, GGCACCCAGCACAATGAA; R, TAGAAGCATTTGCGGTGG.
Mus musculus β-actin: F, CTGTGCCCATCTACGAGGGCTAT; R, TTTGATGTCACGCACGATTTCC. Homo sapiens ACPL2: F, ATGGAGCACTTCAAGGTAA; R, AGCAGAGTAGAGGGCAAA. Homo sapiens RABL4: F, GGAATGGATTTGGTGGTGAA; R, GAGATGCCTGGAGCCTGTGA. Homo sapiens SLC27A3: F, GTTCGGATGGCAAATGAGG; R, TGTACCGGGCAGTTGTGAG. Homo sapiens STAU1 F, ATCCGATTAGCCGACTGG; R, ACTTGAGTGCGGGTTTGG. Homo sapiens β-actin: F, GGCACCCAGCACAATGAA; R, TAGAAGCATTTGCGGTGG.
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