Total RNA were purified from 2 × 105 cultured cells by the ReliaPrep RNA Cell Miniprep System (Promega). cDNA was synthesized from 1 μg total RNA using the GoScript Reverse Transcription System (Promega). Real-time PCR was performed in 15 μl volume with the GoTaq qPCR System (Promega) on the CFX Connect Real-Time PCR Detection System (Bio-Rad). The PCR cycle was run as follows: pre-denaturation by 95°C for 5 min, and then 95°C for 15 s and 60°C for 1 min for 40 cycles. To calculate relative gene expression, the 2–ΔΔCT method was used. Primer sequences for real-time PCR are listed below. To correct for differences in both RNA quality and quantity between samples as well as changes in oxygen exposure, six housekeeping genes were used, namely, beta-actin (actb), glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13a (rpl13a), 45S pre-ribosomal RNA (rn45s), 28S ribosomal RNA (rn28s1), and alpha-tubulin-1 (tuba1):
Gotaq qpcr system
Manufactured by Promega
Sourced in China
The GoTaq qPCR System is a real-time PCR system designed for gene expression analysis and quantification. It includes a thermocycler instrument and associated reagents for performing quantitative PCR (qPCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Reliaprep rna cell miniprep system, by Promega (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Goscript reverse transcription kit, by Promega (1 mentions)
Rnaprep pure tissue kit, by Tiangen Biotech (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Abi 7500 real time pcr system, by Promega (1 mentions)
Rneasy mini purification kit, by Qiagen (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Dnase 1 treated, by Thermo Fisher Scientific (1 mentions)
9 protocols using gotaq qpcr system
1
Gene Expression Analysis of Cultured Cells
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from dissected midguts (50 guts per sample) or brains (150 brains per sample) using RNAprep Pure Tissue Kit (TIANGEN Biotech, Cat# DP431). cDNA was synthesized using GoScript™ Reverse Transcription kit (Promega, Cat# A2790). 0.5 mg total RNA was used for reverse transcription, and the cDNA was diluted 10 times with water and further used in real time PCR. Real time quantitative PCR was performed in at least triplicate for each sample using GoTaq® qPCR System (Promega, Cat# A6001). Expression values were calculated using the ΔΔCt method and relative expression was normalized to RpL23. The expression in control sample was further normalized to 1.
Primer sequences are indicated in Supplementary Table2 .
Primer sequences are indicated in Supplementary Table
3
Quantitative Gene Expression Analysis
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Total RNA was isolated using RNeasy (Qiagen, Germany), and single-strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, Fitchburg, WI) with a random primer. PCR was performed using the Go Taq qPCR system (Promega) at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The PCR primer pairs are described in Supplementary file 1 .
4
Gene Expression Analysis by qPCR
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Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, USA). Complementary DNA was synthesized from total RNA using reverse transcriptase and random primers. Quantitative real-time PCR was performed in the GoTaq® qPCR System (Promega, Beijing, China). The gene-specific primers are as follows: SMYD3, F 5′-GTCTTCAAACTTATGGATGGAGC-3′, 5′-GGCATCCTGTATTTCTTCTCTCA-3′; S1PR1, F 5′-CAGCAAATCGGACAATTCCT-3′; R 5′-GCCAGCGACCAAGTAAAGAG-3′; β-actin, F 5′-CTCCCTGGAGAAGAGCTACG-3′, R 5′-ACAGGACTCCATGCCCAG-3′; and GAPDH, F 5′-GAAGGTGAAGGTCGGAGTCAACG-3′; R 5′-TGCCATGGGTGGAATCATATTGG-3′.
5
Real-Time PCR Using Promega GoTaq
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The real-time PCR experiments were performed according the manufacture protocol (GoTaq qPCR System, Promega, Catalog#A6001) on the ABI 7500 Real-Time PCR System. Each sample were examined in triplicate from three independent experiments. The PCR primers used in this study are as follows:
6
RT-PCR Analysis of Key Cellular Markers
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The RT-PCR experiments were performed using the GoTaq qPCR System (Promega, #A6001) on the ABI 7500 Real-Time PCR System. Each sample was examined in triplicate from three independent experiments. The PCR primers for Romo1, Glut1, Glut3, IL-6, NOS2, TNF-α, Arginase1, Ym1, IL-10 and β-Actin were designed and synthesized by Sangon Biotech. Related primer sequence was provided in Table 2 .
7
Quantitative Gene Expression Analysis
8
Quantitative Gene Expression Analysis
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Cells were treated with AOs as described above. After RNA isolation, 0.5–1 µg of total RNA was DNase I treated (Ambion), reverse transcribed using the Taqman Reverse Transcription Kit (Applied Biosystems), and SYBR-green qPCR was performed using the GoTaq qPCR system (Promega) according to the manufacturers’ recommendations. Expression of target genes was normalized to the expression of the housekeeping gene lysosomal aspartic protease (LAP) and fold changes in expression were calculated using the 2(−ΔΔCT) method (Livak and Schmittgen 2001 (link)).
9
Quantitative RT-qPCR Analysis of Target Genes
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Cells were treated with AOs as described above. After RNA isolation, 0.5-1 µg of total RNA was DNaseI treated (Ambion), reverse transcribed using the Taqman reverse transcription kit (Applied Biosystems), and SYBR-green qPCR was performed using the GoTaq qPCR system (Promega) according to the manufacturers' recommendations. Expression of target genes was normalized to the expression of the housekeeping gene lysosomal aspartic protease (LAP) and fold changes in expression were calculated using the 2 (-ΔΔCT) method (Livak & Schmittgen 2001) .
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