Total RNA was extracted from duodenal, jejunal, and ileal samples using TRIzol Reagent (TaKaRa, Dalian, China). The concentration and purity of total RNA were assayed by spectrophotometer (Beckman Coulter, DU800) at 260 and 280 nm. The ratio of absorption (260/280 nm) of samples was between 1.8 and 2.0. Then, each RNA sample was reverse-transcribed into cDNA using reverse transcriptase (Takara, Tokyo, Japan) after detection of RNA concentration and purity by spectrophotometer (Beckman Coulter, DU800). The PCR primer sequences were designed using Primer Premier 5.0 and are listed in Supplementary Table 1 . Briefly, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA), with a total of 10 μL of assay solution containing 5 μL SYBR Green mix (TaKaRa, Dalian, China), 0.2 μL Rox, 3 μL deionized H2O, 1 μL cDNA template, and 0.4 μL each of forward and reverse primers. The comparative Ct value method was used to quantify mRNA expression relative to β-actin expression (22 (link)).
Quanstudio 6 flex real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuanStudio 6 Flex Real-Time PCR detection system is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qPCR) analysis. It provides accurate and reliable detection and quantification of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green mix, by Takara Bio (4 mentions)
Reverse transcriptase, by Takara Bio (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Du800, by Beckman Coulter (2 mentions)
Reverse transcriptase kit, by Takara Bio (2 mentions)
Rnaiso plus, by Takara Bio (2 mentions)
Sybr green supermix, by Takara Bio (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus reagents, by Takara Bio (1 mentions)
Option monitor 3 real time pcr detection system, by Bio-Rad (1 mentions)
5 protocols using quanstudio 6 flex real time pcr detection system
1
Quantitative Analysis of Intestinal Gene Expression
2
Quantitative PCR Analysis of Gene Expression
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IPEC-J2 cells were harvested and the total RNA was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm [69 (link)]. cDNA was synthesized using a Reverse Transcriptase kit (Takara, Dalian, China). Briefly, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 µL of assay solution containing 5 µL SYBR Green mix (Takara), 0.2 µL Rox, 3 µL deionized H2O, 1 µL cDNA template, and 0.4 µL each of forward and reverse primers (Qingke, Beijing, China). The relative gene expressions compared with the housekeeping gene β-actin were calculated by 2−CT [70 (link)].
3
Quantitative PCR Analysis of Jejunal RNA
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Total RNA was extracted from jejunal samples using TRIzol Reagent (TaKaRa, Dalian, China). Then, each RNA sample was reverse-transcribed into cDNA using reverse transcriptase (Takara, Tokyo, Japan) after detection of RNA concentration and purity by spectrophotometer (Beckman Coulter, DU800). The PCR primer sequences were designed using Primer Premier 5.0 and are listed in Additional file 1 : Table S1. Briefly, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 μL of assay solution containing 5 μL SYBR Green mix (Takara), 0.2 μL Rox, 3 μL deionized H2O, 1 μL cDNA template, and 0.4 μL each of forward and reverse primers. The comparative Ct value method was used to quantify mRNA expression relative to β-actin expression.
4
IPEC-J2 Cell RNA Extraction and qPCR
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IPEC-J2 cells were harvested and the total RNA was extracted using RNAiso Plus (Takara, Dalian, China)
according to manufacturer's instructions. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm [28] . And then cDNA was synthesized using a Reverse Transcriptase kit (Takara, Dalian, China). Brie y, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 µL of assay solution containing 5 µL SYBR Green mix (Takara), 0.2 µL Rox, 3 µL deionized H 2 O, 1 µL cDNA template, and 0.4 µL each of forward and reverse primers (Qingke, China). The relative gene expressions compared with the housekeeping gene β-actin were calculated by 2 - CT [29] .
according to manufacturer's instructions. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm [28] . And then cDNA was synthesized using a Reverse Transcriptase kit (Takara, Dalian, China). Brie y, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 µL of assay solution containing 5 µL SYBR Green mix (Takara), 0.2 µL Rox, 3 µL deionized H 2 O, 1 µL cDNA template, and 0.4 µL each of forward and reverse primers (Qingke, China). The relative gene expressions compared with the housekeeping gene β-actin were calculated by 2 - CT [29] .
5
Quantifying Gene Expression in Small Intestine
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Real-time quantitative PCR was performed in an Option Monitor 3 Real-Time PCR Detection System (Bio-Rad) using the SYBR Green Supermix (TaKaRa). Expression levels of β-actin (housekeeper genes), SGLT1, GLUT2, divalent metal transporter 1 (DMT1), TNF-α, IL-6, ZO-1, Occluding and Claudin-1 in the small intestinal were analysed using SYBR Premix Ex Taq II (Tli RNaseH Plus) reagents (TakaRa) and the QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems). All primers were commercially synthesised and purified by Sangon Biotech Co. Ltd and are shown in Table 2. The reaction was performed in a volume of 10 μl consisting of 5 μl of SYBR Premix Ex Taq (2×), 1 μl of reverse primers, 1 μl of forward primers, 2 μl of doubled-distilled water and 1 μl of cDNA template. Cycling conditions were as follows: 5°C for 30 s, followed by forty cycles at 95°C for 5 s, 60°C for 34 s, under melt curve conditions at 95°C for 15 s, 60°C for 1 min and then 95°C for 15 s (temperature change velocity 0•5°C/s). The target gene mRNA expression level was calculated using the 2 -ΔΔCt method (17) . Each sample was repeated in triplicate.
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