Total RNA was isolated using TRIzol® reagent reversely transcribed into cDNA using Superscript II reverse transcriptase (Toyobo Life Science, Osaka, Japan). The qRT-PCR reaction system contained 2 μL of cDNA sample solution, 10 μL of SYBR-Green PCR master mix, 0.5 μL of forward and reverse primers (1 μM), and 7.5 μL of H2O. The primer sequences were as follows: SIRT1, forward 5′-GCC AGA GTC CAA GTT TAG AAG A-3′, reverse 5′-CCA TCA GTC CCA AAT CCA G-3′; FOXO1, forward 5′-GGC TGA GGG TTA GTG AGC AG-3′ and reverse 5′-AAA GGG AGT TGG TGA AAG ACA-3′ and GAPDH, forward 5′-CCT CAA GAT CAT CAG CAA TG-3′ and reverse 5′-CCA TCC ACA GTC TTC TGG GT-3′. The amplification process was carried out as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 45 s and elongation at 72 °C for 45 s, with the final extension step maintaining at 72 °C for 10 min. qRT-PCR was performed using an ABI Prism 7500 Fast Real-time PCR instrument (Applied Biosystems; Foster City, CA, USA), and analyzed using the 2-ΔΔCt method. The mRNA levels of SIRT1 and FOXO1 were normalized to those of GAPDH to assess the significance of the differences between the groups.
Abi prism 7500 fast real time pcr instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7500 Fast Real-time PCR instrument is a thermal cycler designed for fast and accurate real-time PCR analysis. The instrument uses a 96-well block format and is capable of performing real-time quantitative PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Superscript 3 platinum sybr green one step qrt pcr kit, by Thermo Fisher Scientific (2 mentions)
Rnaprep pure tissue kit, by Tiangen Biotech (2 mentions)
Tag man master mix, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Superscript 2, by Toyobo (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Primescript reverse transcriptase, by Takara Bio (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr fast qpcr mix, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol regent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Bestar qpcr rt kit, by DBI Bioscience (1 mentions)
11 protocols using abi prism 7500 fast real time pcr instrument
1
Quantitative Analysis of SIRT1 and FOXO1
2
Profiling Gene Expression in HUVECs
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Total RNA was extracted from HUVECs by using TRIzol (Life Technologies Corporation, USA). Then, the reverse RNA was conducted by PrimeScript Reverse Transcriptase (Takara, China). The reaction conditions were as follows: 42°C for 2 minutes, 95°C for 5 seconds and 37°C for 15 minutes. The cDNA was subsequently obtained and stored at 4°C for further use. Then 1 μg of RNA samples were selected for quantitative polymerase chain reaction (qPCR), and the obtained cDNA was analyzed 3 times using SYBR green PCR master mix (Fermentas Life Sciences, USA). qRT-PCR was performed by using an ABI Prism 7500 Fast Real-time PCR instrument (Applied Biosystems, USA). The amplification process was carried out as follows: pre-denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 45 seconds. The internal control for microRNAs was U6, and the internal control for MCP-1, ICAM-1, and phosphatase and tensin homolog (PTEN) was β-actin. The 2−ΔΔCt method was applied for the analysis of qRT-PCR results. Each experiment was repeated 3 times.
3
Quantification of Nrf2, HO-1, TNFα, and IL-6 in HUVEC cells
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Total RNA was extracted from HUVEC cells of different groups with Trizol to obtain cDNA by reverse transcription. qRT-PCR was performed to determine the expression levels of Nrf2, HO-1, TNFα, and IL-6 using SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA) in an ABI PRISM 7500 Fast Real-time PCR instrument (Applied Biosystems, Foster City, CA). I Amplification conditions were set as follows: pre-denaturation at 95°C for 2 min, then 40 cycles of 95°C for 20 s and 58°C for 20 s. Human GAPDH gene was used as an internal reference. Specific primers were shown below: Nrf2: forward, 5′-TCC GGG TGT GTT TGT TCC AA-3′, reverse, 5′-CGC CCG CGA GAT AAA GAG TT-3′; HO-1: forward, 5′-CAG GCA ATG GCC TAA ACT TC-3′, reverse, 5′-GCT GCC ACA TTA GGG TGT CT-3′; TNFα: forward, 5′-GAG GCC AAG CCC TGG TAT G-3′, reverse, 5′-CGG GCC GAT TGA TCT CAG C-3′; IL-6: forward, 5′-CCT GAA CCT TCC AAA GAT GGC-3′, reverse, 5′-TTC ACC AGG CAA GTC TCC TCA-3′; GAPDH: forward, 5′-CCT CAA GAT CAT CAG CAA TG-3′, reverse, 5′-CCA TCC ACA GTC TTC TGG GT-3′. The data was analyzed according to the 2–ΔΔCt method. All reactions were performed at least 3 times.
4
Quantification of Peritoneal Tissue Gene Expression
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The peritoneal tissue RNA was extracted with RNAprep pure tissue kit (Tiangen Biotech Co., LTD., Beijing, China). The mRNA of AQP-1, ZO-1, and PPAR-γ was amplified by quantitative real-time RT-PCR. It was carried out using SuperScriptIII Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, California, U.S.A.) by an ABI PRISM 7500 Fast real-time PCR instrument (Applied Biosystems, Foster City, CA, U.S.A.). The qRT-PCR was carried out with the following procedures: 50°C for 3 min, 95°C for 5 min, followed by 38 cycles of 95°C for 15 s, 60°C for 30 s, with specific primers as follows: AQP-1, forward primer, 5′-TGGCCGAAATGACCTGGCTCG-3′, reverse primer, 5′-GGCGCCTCCGGTCAGTGGTA-3′; ZO-1, forward primer, 5′-AGCGAAGCCACCTGAAGATA-3′, reverse primer, 5′-GATGGCCAGCAGGAATATGT-3′; PPAR-γ, forward primer, 5′-TGATATCGACCAGCTGAACC-3′, reverse primer, 5′-GTCCTCCAGCTGTTCGCCA-3′; GAPDH, forward primer, 5′-AATGCATCCTGCA CCACCA A-3′, reverse primer, 5′-GATGCCATAT TCATTGT CATA-3′. The relative mRNA amounts of AQP-1, ZO-1, and PPAR-γ were calculated by comparative Ct method.
5
Quantification of miR-149 Expression in HUVECs
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Total RNA was extracted from HUVECs using TRIzol® reagent (Invitrogen). Template cDNA was synthesized from RNA by reverse transcriptase using ReverTra Ace qPCR RT kit (Toyobo, Tokyo, Japan). The qPCR reaction system was composed of cDNA (2 μL), PCR master mix (10 μL), forward primer (0.5 μL), reverse primer (0.5 μL), and H2O (7.5 μL). The primer sequences were as follows: miR-149, forward 5′- GGC TCT GGC TCC GTG TCT T,′ reverse 5′- CAG TGC AGG GTC CGA GGT ATT-3′; U6, forward 5′-CTC GCT TCG GCA GCA CA-3′, and reverse 5′-AAC GCT TCA CGA ATT TGC GT-3′. All reactions ran the initial denaturation at 95°C for 7 minutes, followed by 40 cycles of denaturation at 95°C for 30 s and annealing at 60°C for 45 s. RT-qPCR was carried out using an ABI Prism 7500 Fast Real-time PCR instrument (Applied Biosystems; Foster City, CA, USA).
6
Lung Tissue RNA Isolation and Analysis
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For Total RNA isolation from lung tissues, TRIzol reagent was used (Invitrogen, Carlsbad, CA). Random primed cDNAs were produced by reverse-transcription of total RNA samples by using High Capacity RNA to cDNA Synthesis kit (Invitrogen, Carlsbad, CA). A real-time PCR analysis was applied with the ABI Prism 7500 Fast Real Time PCR Instrument (Applied Biosystems, CA) using Tag Man Master Mix (Applied Biosystems, CA). Each value was standardized to the levels of GAPDH. The samples were quantified for SP-A (Rn-04338808_g1, Thermo Scientific) and VEGF (Rn01511610_m1, Thermo Scientific), utilizing the comparative Ct (ΔΔCt) method [19 ].
7
Gene Expression Profiling of Paw Tissues
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According to the manufacturer’s total RNA from paws was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was obtained by reverse transcription of total RNA samples with the cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). Real time PCR reactions were performed in triplicate (heated to 50 °C for 2 min followed by 1 cycle of denaturation at 94 °C for 10 min, 40 cycles of 94 °C for 15 s, and 60 °C for 60 s). Standard curves were prepared for target genes and endogenous reference (HPRT) in each sample. The ABI Prism 7500 Fast Real Time PCR Instrument (Applied Biosystems, CA, USA) for real-time PCR analysis was used in this study with using Tag Man Master Mix (Applied Biosystems, CA, USA). All results are standardized according to GAPDH levels. The samples were quantified for MAPK, Fyn, Src, and STAT3 genes using the comparative Ct (ΔΔCt) protocol, according to the Assays–on-Demand User’s Manual (Applied Biosystems, CA, USA).
8
Quantification of Cardiac Gene Expression
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Total RNA was isolated using a TRIzol reagent (Invitrogen) and reversely transcribed to cDNA using the PrimeScript RT Reagent Kit (TaKaRa). Sequences for primers are as follows: NPPA (encoding for ANP) forward: 5′-ACC AAG GGC TTC TTC CTC T-3′, NPPA reverse: 5′-TTC TAC CGG CAT CTT CTC C-3′; β-MHC forward: 5′-TCT GGA CAG CTC CCC ATT CT-3′, β-MHC reverse: 5′-CAA GGC TAA CCT GGA GAA GAT G-3′; and GAPDH forward: 5′-AAC TTT GGC ATT GTG GAA GG-3′, GAPDH reverse: 5′-ACA CAT TGG GGG TAG GAA CA-3′. Real-time PCR was performed with SYBR® Fast qPCR Mix (TaKaRa) using an ABI Prism 7500 Fast Real-time PCR instrument (Applied Biosystems; Foster City, CA, USA). Relative ANP and β-MHC mRNA was determined by the 2-ΔΔCt method. This experiment was repeated three times.
9
Quantitative Analysis of miR-375 and Ctgf Expression
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Total RNA from rat brains and cells was extracted with RNAprep pure tissue kit (Tiangen Biotech Co., Ltd., Beijing, China) and RNeasy Mini Kit (Qiagen, Germantown, MD, U.S.A.), respectively. The expression levels of miR-375 and Ctgf were amplified by quantitative real-time RT-PCR. It was carried out using SuperScriptIII Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, California, U.S.A.) by an ABI PRISM 7500 Fast Real-time PCR instrument (Applied Biosystems, Foster City, CA, U.S.A.). It was carried out with the following procedures: 94°C for 2 min, 94°C for 20 s, followed by 40 cycles of 58°C for 20 s and 72°C for 20 s. The specific primers used in quantitative real-time RT-PCR were displayed as follows: miR-375: forward, 5′-ACACTCCAGCTGGGTTTGTTCGTTCGGCTCGC-3′; reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACGCGA-3′; Ctgf: forward: 5′-CTTCTGCGATTTCGGCTCC-3′; reverse, 5′-TACACCGACCCACCGAAGA-3′; GAPDH: forward, 5′-GGTGGTCTCCTCTGACTTCAACA-3′; reverse, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′; U6: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′- AACGCTTCACGAATTTGCGT-3′. The data were analyzed according to the 2−ΔΔCt method.
10
Quantifying Gene Expression in Ischemic Mice Brains
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Total RNA of the cortex of the ischemia side in mice brains and cultured cells was extracted by using TRIzol Regent (Invitrogen), according to the manufacturer’s instructions. Approximately 2 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using a PrimeScriptTM RT reagent kit (Takara). qPCR assay was performed using SYBR Green PCR Master Mix (Takara) on an ABI PRISM 7500 Fast Real-time PCR instrument (Applied Biosystems). GAPDH and U6 were used as the internal reference for mRNA and miRNAs, respectively. The relative expression fold of targets was calculated using the 2−ΔΔCt method. The primers used were as follows: miR-9-3p forward: 5ʹ-GAGGCCCGTTTCTCTCTTTG-3ʹ, reverse: 5ʹ-AGCTTTATGACGGCTCTGTG-3ʹ; U6 forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ; FGF19 forward: 5ʹ-CGCTGTCGGTAGCCAGAG-3ʹ, reverse: 5ʹ-CTCTGCACGCCCTTGATG-3ʹ; HO-1 forward: 5ʹ-GGAACTTTCAGAAGGGCCAG-3ʹ, reverse: 5ʹ-GTCCTTGGTGTCATGGGTCA-3ʹ; NQO-1 forward: 5ʹ-GTATCCTGCCGAGTCTGTT-3ʹ, reverse: 5ʹ-GATCCCTTGCAGAGAGTACA-3ʹ; GAPDH forward: 5ʹ-CCACCCATGGCAAATTCCATGGCA-3ʹ, reverse: 5ʹ-TCTAGACGGCAGGTCAGGTCCACC-3ʹ.
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