Cellular RNA was extracted using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Crawley, UK). The RNA was quantified using a Nanodrop ND1000 spectrophotometer (Labtech, Ringmer, UK), and 1 μg of total RNA was added to a genomic DNA elimination reaction, followed by conversion to cDNA (Quantitect Reverse Transcription Kit, Qiagen), according to the manufacturer's instructions. Quantitative PCR was performed using exon–exon junction spanning primers (Supplemental Table S2 ) and the IQ5 system (Bio-Rad, Hemel Hempstead, UK). The starting quantity of mRNA was determined using standard curves generated from serial dilutions of pooled reference RNA with Quantifast SYBR green (Qiagen). The target and reference genes were analyzed in triplicate, and mRNA expression normalized to the ACTB and RPL19 reference genes (Supplemental Table S2 ) using the IQ5 system (Bio-Rad), with inter-run correlation and run-dependent differences corrected using qBase software on the IQ5 system (Bio-Rad), as described previously [52 (link)]. The reference genes did not differ in their expression with the treatments, and the reference genes were amplified with the same efficiency as the target genes.
Iq5 system
Manufactured by Bio-Rad
Sourced in United States, Japan, China, Canada, Spain
The IQ5 system is a real-time PCR detection system manufactured by Bio-Rad. It is designed for quantitative gene expression analysis and detection of nucleic acids. The system utilizes optical detection technology to monitor and analyze PCR reactions in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (55 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
Sybr premix ex taq 2, by Takara Bio (14 mentions)
Primescript rt reagent kit, by Takara Bio (13 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (12 mentions)
Iscript cdna synthesis kit, by Bio-Rad (9 mentions)
Sybr green 1, by Takara Bio (9 mentions)
Goscript reverse transcription system, by Promega (8 mentions)
Rnaiso plus, by Takara Bio (7 mentions)
Sybr green pcr master mix, by Bio-Rad (7 mentions)
Mirneasy mini kit, by Qiagen (6 mentions)
Trizol reagent, by Takara Bio (6 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Iq sybr green supermix, by Bio-Rad (5 mentions)
Sybr premix ex taqtm 2, by Takara Bio (5 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (5 mentions)
Nucleospin rna kit, by Macherey-Nagel (4 mentions)
Sybr green mix, by Bio-Rad (4 mentions)
Faststart universal sybr green master, by Roche (4 mentions)
237 protocols using iq5 system
1
Quantitative RT-PCR Protocol for mRNA Expression
2
Quantifying Gene Expression in PBLs
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Total cellular RNA was extracted from peripheral blood cells (PBLs) from each patient following the clinical trial, using a Nucleo Spin RNA kit (Macherey-Nagel, Düren, Germany). One microgram of total RNA was reverse-transcribed in a final volume of 20 μl with the First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) using random primers. The SYBR Green PCR system (BioRad, Hercules, CA, USA) was used to perform real-time PCR with an iQ5 system (Biorad, Spain). The sequences of the oligonucleotides used corresponded to the following shown in Table 1 :
Primers used for qPCR amplications
| GENE | Forward | Reverse |
|---|---|---|
| 18S | 5′- CTCAACACGGGAAACCTCAC - 3′ | 5′-CGCTCCACCAACTAAGAACG - 3′. |
| BAX | 5′- CACTCCCGCCACAAAGAT - 3′ | 5′- CAAGACCAGGGTGGTTGG - 3′ |
| EPO | 5′ - TGTTTTCGCACCTACCATCA - 3′ | 5′ - AAGTCACAGCTTGCCACCT - 3′ |
| SOX2 | 5′ – GGGGGAATGGACCTTGTATAG - 3′ | 5′ – CGCTCCACCAACTAAGAACG - 3′ |
| OCT4 | 5′- CTTCGGATTTCGTCTTCTCG-3′ | 5′- CTTAGCCAGGTCCGAGGAT - 3′ |
3
Comprehensive RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted from tissues and cells with Trizol reagent (TaKaRa, Otsu, Japan) according to the specifications of the manufacturer. NanoDrop 2000 (Thermo, USA) was used to measure RNA concentration and reverse transcription kits (TakaRa) were used to obtain cDNA from RNA. SG (SYBR green kits) and a Bio-Rad iQ™5 system (Bio-Rad, USA) were used for real-time quantitative PCR following the specifications of the manufacturer. Mouse GAPDH was regarded as a housekeeping gene. We used a specific procedure and primer for the miRNA experiment. Mouse U6 was regarded as a housekeeping gene for miRNA. The levels of miRNAs were quantified by qRT-PCR using Bulge-Loop™ miRNA qRT-PCR Primer Sets (RiboBio, Guangzhou, China) and analysis performed on a Bio-Rad iQ™5 system (Bio-Rad). The primers for miR-129-5p and U6 small nuclear RNA were obtained from RiboBio Company (Guangzhou, China) and the sequences are covered by a patent. The primer sequences used for RT-qPCR analyses are listed in Table 1 .
Primer sequences used for real-time qPCR.
| Name | Forward | Reverse |
|---|---|---|
| Cyclin E | CAGAGCAGCGAGCAGGAGC | GCAGCTGCTTCCACACCACT |
| Cyclin D | TAGGCCCTCAGCCTCACTC | CCACCCCTGGGATAAAGCAC |
| Cyclin B | AACTTCAGCCTGGGTCG | CAGGGAGTCTTCACTGTAGGA |
| P27 | TGGGTCTCAGGCAAACTCT | TGGGTCTCAGGCAAACTCT |
| G3BP1 | AACTCTTCCTATGCCCACGG | GTAGCCCCATCACTTGGACC |
| CDK6 | TGGATAAAGTTCCAGAGCCCG | TTCTGCGGTTTCAGATCACGA |
| GAPDH | TGCTGAGTATGTCGTGGAGTCT | ATGCATTGCTGACAATCTTGAG |
4
Gene Expression Analysis of S. Typhimurium
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The S. Typhimurium SL1344 suspension was illuminated by 405 nm LED apparatus at 4 °C for 240 min, and non-illuminated (control) cells were incubated at 7 °C for 240 min. After incubation, each sample was centrifuged (5000× g, 5 min, 4 °C) and resuspended in PBS. Total RNA was extracted using a Bacterial RNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. RNA integrity and concentration were measured using a spectrophotometer (Nano-200; Aosheng Instrument Co., Ltd., Hangzhou, China). The RNA was then immediately reverse-transcribed to complementary DNA using the Takara PrimeScript RT Reagent Kit (Takara, Kyoto, Japan). Real-time polymerase chain reaction was conducted with the IQ5 system (Bio-Rad Laboratories, Hercules, CA, USA). The 16S rRNA gene was used to normalize the gene expression levels. The sequences of the primers are listed in Table S1 . The transcription of target genes was analyzed with the −1/2–ΔΔCt method [16 (link)].
5
Esophageal Tight Junction Protein Expression
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Total RNA was extracted from esophageal biopsy sections using an RNA easy mini kit (Qiagen Sciences, Ontario, Canada) and treated with RNAse-free DNase set (Qiagen Sciences, Ontario, Canada) to remove contaminating DNA (cDNA). cDNA synthesis was performed using an oligo-dT primer and M-MLV reverse transcriptase (Invitrogen, Ontario, Canada). The cDNA samples were used to measure CLDN-2, CLDN-3, and ZO-1 by real-time quantitative PCR using SsoFast™ EvaGreen Supermix (Bio-rad, Ontario, Canada) and iQ5 system (Bio-rad, Ontario, Canada). The primers are described in Supplementary Table 3; GAPDH gene was used as housekeeping gene. The PCR was performed at 95°C for 30 s, followed by 30 cycles (ZO-1 and GAPDH) or 36 cycles (CLDN-2) or 38 cycles (CLDN-3) at 95°C for 6 s, and 60°C for 10 s; then a melt curve analysis from 55–95°C (in 0.5°C increments) was conducted. The PCR efficiencies were measured for each primer pair. The analysis of relative gene expression levels was completed by the 2-ΔΔCT method. Primer sequences and product sizes used for determination of the mRNA expression of TJ proteins are shown in Supplementary Table 3.
6
Quantitative RT-PCR Analysis of Inflammatory Cytokines
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Total RNA was extracted from tissues using TRizol reagent (Invitrogen). RNA was subjected to reverse transcription with reverse transcriptase as the manufacturer's instructions (Fermentas). Quantitative real-time PCR was performed using the Bio-Rad iQ5 system, and the relative gene expression was normalized to internal control as Beta actin. Primer sequences for SYBR Green probes of target genes are as follows, IL-1β
: CTGGTGTGTGACGTTCCCATTA and CCGACAGCACGAGGCTTT; IL-6: TTCCATCCAGTTGCCTTCTTG and TTGGG AGTGGTATCCTCTGTGA; Tnf-α: CATCTTCTCAAAATTCGAGTGACA and TGG GAGTAGACAAGGTACAACCC.
: CTGGTGTGTGACGTTCCCATTA and CCGACAGCACGAGGCTTT; IL-6: TTCCATCCAGTTGCCTTCTTG and TTGGG AGTGGTATCCTCTGTGA; Tnf-α: CATCTTCTCAAAATTCGAGTGACA and TGG GAGTAGACAAGGTACAACCC.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from tissues using Trizol reagent (Invitrogen). RNA was subjected to reverse transcription with reverse transcriptase as per manufacturer's instructions (Fermentas). Quantitative real-time PCR was performed using the Bio-Rad iQ5 system, and the relative gene expression was normalized to internal control as beta-actin. Primer sequences for SYBR Green probes of target genes are as shown in Table 1 .
8
Adipose Tissue RNA Extraction and Analysis
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Total RNA was extracted from adipose tissues using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. cDNA was synthesized using a PrimeScript RT reagent Kit (Takara). Quantitative PCR (qPCR) involved use of UltraSYBR Mixture (CWBiotech) with a Bio-Rad iQ5 system. The 2-ΔΔCTmethod was used to assess relative mRNA expression levels. qPCR primers were in Supplemental Table 3 .
9
Quantifying Gene Expression in Cells
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Total RNA was extracted with Trizol reagent (Invitrogen) according to the manufacturer's protocol and 2 μg was reversed‐transcribed using the GoScript™ Reverse Transcription system (Promega, Madison, WI, USA). For real‐time quantitative PCR, the iQ5 system (Bio‐Rad, Hercules, CA, USA) with SYBR Green I (Takara, Kusatsu, Japan) was used. Amplification was performed at 95°C for 5 minutes, 95°C for 45 seconds and 57°C for 45 seconds of each step for 45 cycles. The housekeeping gene GAPDH was used as a control. The primers used are shown below: Tumour necrosis factor‐α (TNF‐α): Forward, tcttctcattcctgcttgtgg; Reverse, ggtctgggccatagaactga; inducible nitric oxide synthase (iNOS): Forward, gggctgtcacggagatca; Reverse,ccatgatggtcacattctgc; arginase‐1 (Arg‐1):Forward, aaagctggtctgctggaaaa; Reverse, acagaccgtgggttcttcac.
10
Quantitative RT-PCR Analysis of Target mRNA
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Total RNA was extracted from NP cells using Trizol reagent (DingGuo, China). This purified, DNA-free RNA was converted to cDNA using a ReverTraAce qPCR RT Kit (TOYOBO, Japan). Reactions were set up in triplicate in 96-well plates using SYBR Green PCR Master Mix (TIANGEN, China). RT-qPCR analyses were performed to detect target mRNA expression using a real-time IQ5 system (Bio-Rad, West Berkeley, CA, USA), and β-actin was used as an internal control. The total volume of the reaction mixture used for each PCR was 20 μl, which included 10 μl of SYBR Green mixture, 0.6 μl of each primer (10 μmol l−l), 2 μl of cDNA template and 6.8 μl of ddH2O. The following PCR conditions were used for the experiments: 95 °C for 15 min, 95 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s for 40 cycles. Relative gene-expression levels were normalized to β-actin expression levels, and the data were presented as fold changes using the formula 2−ΔΔCT, as recommended by the manufacturer. The sequences of the primers used herein are listed in Table 2 .
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