Under a dissection microscope, the whole aorta was dissected carefully by removing the adipose tissue and collecting all the layers of the aorta. Aortic tissues were homogenized in TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Total RNA was extracted from the aorta and 1 μg of total RNA was reverse transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA), according to the manufacturer's instructions. Real-time PCR was performed using Assay-on-Demand TaqMan probes and primers specific for chemokine receptor-2 (CCR2), IL-17, IL-12p40, IL-6 and GAPDH (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 7500 Sequence Detection System. The level of mRNA expression was normalized with respect to the level of GAPDH mRNA expression.
Amv rt taq polymerase
Manufactured by Promega
Sourced in United States
AMV-RT Taq polymerase is a thermostable DNA polymerase enzyme that combines the properties of Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT) and Thermus aquaticus (Taq) DNA polymerase. This enzyme can be used for both reverse transcription and DNA amplification in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (7 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Sybr green method, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Abi 7500 thermocycler, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman probes and primers, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Lonza (1 mentions)
9 protocols using amv rt taq polymerase
1
Aortic Gene Expression Analysis
2
Profiling Cytokine mRNA Expression in UUO Kidney Model
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Total RNA was extracted from kidney tissues harvested from mice 1 or 2 weeks after induction of UUO. Cytokine mRNA concentrations were assayed by polymerase chain reaction (PCR). Briefly, total RNA was isolated from kidneys using an RNeasy® kit (Qiagen GmBH, Hilden, Germany), and 1 μg of total RNA was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA). Real-time PCR was performed using Assay-on-Demand TaqMan® probes and primers for FSP-1, TGF-β1, TGF-β2, vWF, CD31, VE-cadherin, c-kit, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) on an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems). The levels of mRNA expression for each cytokine were normalized to the level of GAPDH mRNA in the same sample.
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from the cells and tissues, and the mRNA levels of the target genes were assayed by qRT- PCR. Briefly, total RNA was extracted from the primary cultured GECs and 5/6 nephrectomized kidney tissues using the RNeasy kit (Qiagen GmbH), and 500 ng of total RNA was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega). qRT- PCR was performed using either Assay-on-Demand TaqMan probes or the SYBR Green method and primers for PROS1, LGALS1, NFKB1, P53, FN1, and GAPDH (Applied Biosystems) and analyzed using the Applied Biosystems PRISM 7500 sequence detection system. Data were normalized to GAPDH and presented as fold increase compared with RNA isolated from the control group using the 2−ΔΔCT method (28 (link)). All experiments were performed in triplicate. PCR primers used for qRT-PCR are listed in supplemental Table S1 .
4
Quantifying Renal TG2 Expression in UUO Mice
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Total RNA isolated from kidney tissues of unilateral ureteral obstruction (UUO) mice 3, 7, and 14 days after UUO model establishment was subjected to real-time PCR to analyze TG2 mRNA levels. RNeasy mini kit® (Qiagen, Hilden, Germany) was used for total RNA extraction. Reverse transcription was performed using oligo-dT primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA). Real-time PCR was carried out on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, USA) using the Assay-on-Demand SYBR Green method. GAPDH mRNA levels were used for normalization. The sequences of the forward and reverse primers used in the study were as follows: GAPDH forward: 5′-TATGTCGTGGAGTCTACTGGT-3′, GAPDH reverse: 5′-GAGTTGTCATATTTCTCGT-3′, TG2 forward: 5-′GGACATCACCCATACCTACAAG-3′, TG2 reverse: 5′-TCTCTGCCAGTTTGTTCAGG-3′.
5
Quantitative Gene Expression Analysis in Mouse Kidney
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Briefly, 1 μg of total RNA extracted from mouse kidney tissues using the RNeasy Kit (Qiagen, Hilden, Germany) was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA). qPCRs were run using Assay-on-Demand TaqMan probes and primers targeting LPCAT2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems). After normalization to GAPDH expression, mRNA levels were calculated using the comparative Ct method (2–ΔΔCt).
6
Quantifying Gene Expression in Renal Epithelial Cells
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Total RNA was extracted from PTECs, and the mRNA levels of the target genes were assayed by real-time quantitative PCR. Briefly, total RNA was isolated from PTECs using an RNeasy kit (Qiagen GmbH, Germany), and 500 ng of total RNA was reverse-transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, WI, USA). Real-time qPCR was conducted on an ABI PRISM 7500 sequence detection system using either Assay-on-Demand TaqMan probes and primers (for E-cadherin, fibronectin, collagen 1, αSMA and GAPDH) or the SYBR Green method (for GAPDH, TNF-α, IFN-γ, IL-6 primer sequences are available in Table 1 ) (Applied Biosystems, CA, USA). Relative quantification was performed using the 2−∆∆CT method. GAPDH served as a loading control44 (link). All experiments were completed in triplicate.
Sequences of real time PCR primers.
| Target names | Species | Sense (5′-3′) | Antisense |
|---|---|---|---|
| GAPDH | MS | 5′-TATGTCGTGGAGTCTACTGGT-3′ | 5′-GAGTTGTCATATTTCTCGT-3′ |
| TNF-α | MS | 5′-GGGACAAGGCTGCCCCGACT-3′ | 5′-TCCTTGGGGCAGGGGCTCTT-3′ |
| IFN-γ | MS | 5′-AACGCTACACACTGCATCTTGG-3′ | 5′-GCCGTGGCAGTAACAGCC-3′ |
| IL-6 | MS | 5′-GTGCTCCTGGTATTGCTGGT-3′ | 5′-GGCTCCTCGTTTTCCTTCTT-3′ |
7
Quantitative PCR Analysis of Peritoneal Gene Expression
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Total RNA was extracted from the peritoneum, and the mRNA levels of target genes were assayed by real‐time quantitative PCR. Briefly, total RNA was isolated from the peritoneum using the RNeasy kit (Qiagen GmBH), and 500 ng of total RNA was reverse‐transcribed using oligo‐d(T) primers and AMV‐RT Taq polymerase (Promega). Real‐time qPCR was conducted on an ABI PRISM 7500 sequence detection system using either Assay‐on‐Demand TaqMan probes and primers (fibronectin, ST2) or the SYBR Green method (for GAPDH, fibronectin, periostin, TGF‐β, NF‐κB, Nrf‐2, MCP‐1, collagen 1, Bax, BCL2, P53 primer sequences are available in Table S1 ) (Applied Biosystems). Relative quantification was performed with the 2‐∆∆CT method. GAPDH was used as a loading control. All experiments were completed in triplicate.
8
Quantifying Target Gene Expression in hPGECs
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Total RNA was extracted from the cells, and mRNA levels of the target genes were assessed using qRT-PCR. Briefly, total RNA was extracted from hPGECs using an RNeasy kit (Qiagen GmbH, Hilden, Germany), and 50 ng of total RNA were reverse-transcribed using oligo-d (T) primers and AMV-RT Taq polymerase (Promega, Madison, WI, USA). qRT-PCR was performed using the SYBR Green method and primers for alpha-smooth muscle actin (αSMA), KLF4, KLF2, transforming growth factor (TGFβ), AT1R, angiotensin type-2 receptor (AT2R), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems, Foster City, CA, USA). The PCR data were analyzed using the Applied Biosystems PRISM 7500 sequence detection system. Relative quantification was performed using the 2−ΔΔCT method [57 (link)]. GAPDH was used as a loading control, and mRNA expression levels were normalized to those of GAPDH. All the experiments were performed in triplicate. The PCR primers used for qRT-PCR are listed in Table 1 .
9
Periostin Regulates Collagen Expression
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Human collecting duct cells (H5 cell line) were cultured at 37 ° C in a 5% carbon dioxide atmosphere in Dulbecco's Modified Eagle's Medium/F-12 1: 1 mixture with HEPES, L-glutamine (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum, 5 mg/ml apotransferrin, 3.10-4 M sodium selenite, 10 mg/ml insulin from bovine pancreas and 10-3 M dexamethasone (cell line and protocol were provided by Remi Piedagnel). Cells were grown to approximately 80% confluence and subjected to serum deprivation (1% FBS) for 24 h before experimental cell treatment. The H5 cells were treated with 10 ng TGF-β (positive control) or 0.25, 0.5 or 1.0 μg periostin for 24 h. The TGF-β and periostin were purchased from R&D systems. Total RNA was isolated from cells using the RNeasy ® kit (Qiagen GmBH, Hilden, Germany), and 1 μg of total RNA was reverse transcribed using oligo-d(T) primers and AMV-RT Taq polymerase (Promega, Madison, Wis., USA). Real-time polymerase chain reaction was performed using assay-on-demand TaqMan ® probes and primers for type I, alpha 1 collagen (collagen Ia1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Applied Biosystems) and an ABI PRISM ® 7500 Sequence Detection System (Applied Biosystems). The levels of mRNA expression for collagen Ia1 were normalized with respect to the level of GAPDH mRNA expression.
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