The messenger RNA (mRNA) levels of tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and TMUB1 were determined by qRT‐PCR. Total RNAs were prepared using TRIpure (BioTeke, China) and reverse‐transcribed using BeyoRT II M‐MLV reverse transcriptase (Beyotime, China). Quantitative PCR was performed using SYBR Green dye (Solarbio, China) to measure amplification on a fluorescence quantitative PCR instrument (Exicycler 96, Bioneer). The sequences of primers were as follows (5′–3′): IL‐6 forward (mus): ATGGCAATTCTGATTGTATG, and reverse (mus): GACTCTGGCTTTGTCTTTCT; TMUB1 forward (mus): GTAGGCGATGAGGTGACTGT, and reverse (mus): GCTGTGCTGGTGTTGTGG; TNF‐α forward (mus): CAGGCGGTGCCTATGTCTCA, and reverse (mus): GCTCCTCCACTTGGTGGTTT; TMUB1 forward (Homo): CCTCGTGCTACGGCTGAAA, and reverse (Homo): GTTGGGAGGGAGGTGAAGG; IL‐6 forward (homo): GTCCAGTTGCCTTCTCCC, and reverse (Homo): GCCTCTTTGCTGCTTTCA; TNF‐α forward (Homo): GAGTGACAAGCCTGTAGCC, and reverse (Homo): AAGAGGACCTGGGAGTAGAT.
Beyort 2 m mlv reverse transcriptase
Manufactured by Beyotime
Sourced in China
BeyoRT II M-MLV reverse transcriptase is a recombinant enzyme that catalyzes the synthesis of complementary DNA (cDNA) from an RNA template. It is derived from the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase and is suitable for use in various RNA-based applications, such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Solarbio (23 mentions)
Taq pcr mastermix, by Solarbio (11 mentions)
Tripure reagent, by BioTeke (7 mentions)
Tripure, by BioTeke (6 mentions)
Nano 2000, by Thermo Fisher Scientific (5 mentions)
Tripure lysate, by BioTeke (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Exicycler 96, by Bioneer (3 mentions)
Rnase inhibitor, by BioTeke (3 mentions)
Total rna isolation kit, by Tiangen Biotech (3 mentions)
Exicycler 96 real time pcr system, by Bioneer (3 mentions)
Nano 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Exicyclertm 96, by Bioneer (2 mentions)
Sybr green kit, by Solarbio (2 mentions)
Nano 2000 spectrophotometry, by Thermo Fisher Scientific (2 mentions)
Exicyclertm 96 real time pcr system, by Bioneer (2 mentions)
Sybr green 1, by Solarbio (2 mentions)
Tripure kit, by BioTeke (2 mentions)
Trizol, by BioTeke (2 mentions)
Pcr mastermix, by Solarbio (2 mentions)
42 protocols using beyort 2 m mlv reverse transcriptase
1
Quantifying mRNA Levels in Inflammatory Responses
2
Quantitative RT-PCR Analysis of Cartilage Markers
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Total RNA was extracted and reverse transcribed into cDNA by BeyoRT II M-MLV reverse transcriptase (Beyotime). cDNA was subjected to qRT-PCR using SYBR green Kit (Solarbio, Beijing, China). The constructed PCR reaction system was put into ExicyclerTM96 (BIONEER, Daejon, Korea). The relative gene expression values were calculated using the 2−ΔΔCt method. GAPDH was used as internal control. The primers are listed in Table 1 .Table 1
qRT-PCR primers.
| Name | Sequence (5′-3′) |
|---|---|
| OSTF1 forward | TTTACTCAGCCGAATGTG |
| OSTF1 reverse | TCTTCTTCAGGAGCGATG |
| TNF-α forward | CGGAAAGCATGATCCGAGAT |
| TNF-α reverse | AGACAGAAGAGCGTGGTGGC |
| IL-6 forward | CAGCCACTGCCTTCCCTA |
| IL-6 reverse | TTGCCATTGCACAACTCTTT |
| aggrecan forward | CGCCCATCATCAGAAACC |
| aggrecan reverse | TCCAGGCAGCGTAGAGC |
| collagen-Ⅱ forward | AGAGCGGAGACTACTGGATTG |
| collagen-Ⅱ reverse | TCTGGACGTTAGCGGTGTT |
| MMP1 forward | AAAGGCAGGTTCTACATTCGT |
| MMP1 reverse | CTAACTTCATAAGCAGCATCA |
| MMP13 forward | GCCAGAACTTCCCAACCA |
| MMP13 reverse | ACCCTCCATAATGTCATACCC |
3
Quantitative RT-PCR for Gene Expression
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Quantitative RT-PCR was performed to detect the mRNA level of genes. Total RNA was extracted from cells using TriPure reagent, chloroform, and isopropanol following the manufacturer’s instructions. For mRNA detection, RNA was reverse transcribed by using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China), and quantitative RT-PCRs were conducted using gene-specific primers, SYBR Green (Solarbio), and 2 × Taq PCR Master Mix. Quantitative normalization was performed using the expression of β-actin. Relative mRNA expression was calculated using the 2−ΔΔCT method. Primer sequences were provided in Supplementary Table 1 .
4
Quantitative Real-Time PCR for Knockdown Efficiency
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Quantitative real-time PCR (qRT-PCR) was used to determine the relatively higher knockdown efficiency of shRNA and siRNA for further experiments. Total RNA was extracted from OS cells using TRIpure Reagent (Bioteke, Beijing, China). The BeyoRT II M-MLV Reverse Transcriptase (Beyotime Biotechnology, Shanghai, China) and RNase inhibitor (Bioteke, Beijing, China) were used for reverse transcription. The 2×Taq PCR MasterMix and SYBR Green (Solarbio, Beijing, China) were employed to carry out the qRT-PCR assay. In order to normalize lncRNA and mRNA expression, β-actin was used as an endogenous control. 2−ΔΔCt was used to calculate the relative expression level of the target RNA. Table S3
5
Quantitative Analysis of Tight Junction Genes
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Total RNA was extracted from nasal mucosa tissues or cells by employing an RNA Simple Total RNA Kit (Tiangen, Beijing, China). The cDNA synthesis was performed by a BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China). 2×Taq PCR MasterMix (Solarbio) and SYBR Green (Solarbio) were used to quantify the expression of genes. The relative expression of mRNA was calculated by 2 –ΔΔCtmethod. GAPDH was used as the internal control. The sequences of primer were shown in Table 2 .
The Sequences of Primer Used in qRT-PCR
| Genes | Forward Sequences (5’-3’) | Reverse Sequences (5’-3’) |
|---|---|---|
| MAFB (mus) | TCACCTGGAGAACGAGAAGACG | AAGCCGGAGTTGGCGAGT |
| Occludin (mus) | CCTGGAGGTACTGGTCT | ATCTTTCTTCGGGTTTT |
| ZO-1 (mus) | TGCCTCGAACCTCTACTC | GTGGTGGAACTTGCTCAT |
| GAPDH (mus) | TGTTCCTACCCCCAATGTGTCCGTC | CTGGTCCTCAGTGTAGCCCAAGATG |
| MAFB (homo) | AGCACCACCTGGAGAATGAGA | AAGCCGGAGTTGGCGAGT |
| ZO-1 (homo) | CCAGTCCCTTACCTTTCG | CTGCCTCATCATTTCCTC |
| Occludin (homo) | CCTCTTGAAAGTCCACCTC | GCCTACACTACCTCCTAAAA |
| GAPDH (homo) | GACCTGACCTGCCGTCTAG | AGGAGTGGGTGTCGCTGT |
6
Quantitative Analysis of USP43 Expression
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Total RNA was isolated using a TRIpure commercial kit (BioTeke Corporation). RNA concentrations were determined with the NANO 2000 spectrophotometry (Thermo Fisher Scientific). cDNA was synthesized using reverse transcription with a BeyoRT II M-MLV reverse transcriptase (Beyotime Biotechnology). qPCR was performed with SYBR Green (Solarbio Science) using an ExicyclerTM 96 Real-Time PCR System (Bioneer Corporation). Gene expression was normalized to the expression of GAPDH by the 2−ΔΔCt method. The PCR primer sequences are as follows: USP43 F, 5’-AGGGCTTGAAGAACCACG-3’; USP43 R, 5’-CAGCAACCAGAGCAGGAA-3’.
7
RNA Extraction and RT-qPCR Analysis of Lung Cell Cytokines
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Total RNA from the lung tissues and RAW264.7 cells was abstracted by TRIpure lysis (RP1001, BioTeke, Beijing, China). The cDNA was generated with BeyoRT II M-MLV Reverse Transcriptase (D7160L, Beyotime, Shanghai, China). The relative expression of mRNA was evaluated by RT-qPCR using SYBR Green (SY1020, Solarbio, Beijing, China) in Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea). The specific primers were as follows: IL-4, 5’-CATCCTGCTCTTCTTTCTCG-3’, 5’-CCTTCTCCTGTGACCTCGTT-3’; IL-5, 5’-GGCTTCCTGTCCCTACT-3’, 5’-CTTCCATTGCCCACTCT-3’; IL-13, 5’-TTGCCTTGGTGGTCTCG-3’, 5’-CAATATCCTCTGGGTCCTGT-3’; chitinase-like 3 (YM1), 5’-ACTGGAATTGGTGCCCCTAC-3’, 5’-GAGCCACTGAGCCTTCAACT-3’; mannose receptor C-type 1 (MRC1), 5’-AGTGATGGTTCTCCCGTTTC-3’, 5’-TGGGCTCAGGTAGTAGTGTTTT-3’. GAPDH was used as an endogenous control.
8
Quantitative PCR Analysis of Lung Gene Expression
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Using the Total RNA Isolation Kit (Tiangen Biotech Co. Ltd., Beijing, China) and BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China), total RNA was isolated from the lungs and reverse transcribed into the rst cDNA. A quantitative PCR assay was carried out by Exicycler TM 96 Real-time PCR System (Bioneer Corporation, Daejeon, Korea) using SYBR Green (Solarbio, Beijing, China). The level of mRNA was normalized to GAPDH. The primers were synthesized by Genscript Biotechnology Co., Ltd. (Nanjing, China), and the sequences were shown in Table 1.
9
Quantifying TMED10 mRNA Expression
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Total RNA was isolated from brain tissues and primary cortical neurons using TRIpure lyase (Bioteke, Beijing, China). The RNA samples obtained were reverse transcribed into corresponding
cDNA using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China) according to the manufacturer’s instructions. SYBR Green (Solarbio, Beijing, China) and 2 × Taq PCR Master Mix
(Solarbio) were used to detect relative mRNA expression. The primer sequence of TMED10 is as follows: TMED10 F 5′-TCTGTATGCCAAAGAGGATG-3′, TMED10 R 5′-CAATGGACTCGGAAAGGT-3′. The relative
mRNA expression was standardized by β-actin, F 5′-CTGTGCCCATCTACGAGGGCTAT-3′, R 5′-TTTGATGTCACGCACGATTTCC-3′.
cDNA using BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China) according to the manufacturer’s instructions. SYBR Green (Solarbio, Beijing, China) and 2 × Taq PCR Master Mix
(Solarbio) were used to detect relative mRNA expression. The primer sequence of TMED10 is as follows: TMED10 F 5′-TCTGTATGCCAAAGAGGATG-3′, TMED10 R 5′-CAATGGACTCGGAAAGGT-3′. The relative
mRNA expression was standardized by β-actin, F 5′-CTGTGCCCATCTACGAGGGCTAT-3′, R 5′-TTTGATGTCACGCACGATTTCC-3′.
10
Quantification of IL-17A and IL-10 in Mouse Tissues
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RNA was extracted from liver and spleen tissues of mice. RNA concentration was determined under ultraviolet spectrophotometer NANO 2000 (Thermo Fisher Scientific, Pittsburgh, PA, USA). CDNA
was obtained by beyoRT II M-MLV reverse transcriptase (Beyotime). Finally, cDNA was quantified under ExicycleTM96 fluorescence quantification instrument (Bioneer, Daejeon, Korea). The
quantitative results were calculated by 2-ΔΔCt method. The primer sequences used in this study were as follows: MUS IL-17A-F: 5′-AAACACTGAGGCCAAGGAC-3′; MUS
IL-17A-R: 5′-CGTGGAACGGTTGAGGTAG-3′; MUS IL-10-F: 5′-TTAAGGGTTACTTGGGTTGC-3′; MUS IL-10-R: 5′-GAGGGTCTTCAGCTTCTCAC-3′.
was obtained by beyoRT II M-MLV reverse transcriptase (Beyotime). Finally, cDNA was quantified under ExicycleTM96 fluorescence quantification instrument (Bioneer, Daejeon, Korea). The
quantitative results were calculated by 2-ΔΔCt method. The primer sequences used in this study were as follows: MUS IL-17A-F: 5′-AAACACTGAGGCCAAGGAC-3′; MUS
IL-17A-R: 5′-CGTGGAACGGTTGAGGTAG-3′; MUS IL-10-F: 5′-TTAAGGGTTACTTGGGTTGC-3′; MUS IL-10-R: 5′-GAGGGTCTTCAGCTTCTCAC-3′.
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