Transcript levels were analyzed in a CFX Connect™ 96 Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, CA, USA). RT-PCR amplifications were conducted in a 15 µL reaction volume mixture containing 1.5 µL of cDNA, 4 µL ddH2O, 1 µL of 10 pmol forward (sense) primer, 1 µL of 10 pmol reverse (antisense) primer, and 7.5 µL iTaq™ Universal SYBER® Green Supermix (Bio-Rad Laboratories GmbH, CA, USA). Each reaction for each gene was performed in triplicate following PCR protocol: 5 min 94 °C, 30 s 94 °C, 5 s 65 °C, 10 s 75 to 95 °C for melting curve (30 cycles). PCR amplification was performed with different cycles to ensure a linear response in the PCR. Primers used for RT-PCR are shown in Table 2 , and their specificity was checked by separating the PCR products on 1.8% agarose gels. The TaACTIN (AB181991.1) gene was used as a reference gene, and the expression levels of genes were calculated by using the 2−ΔΔCT method [78 (link)].
Cfx connect 96 real time pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The CFX Connect 96 real-time PCR detection system is a lab equipment product designed for real-time PCR analysis. It features a 96-well format and can detect multiple fluorescent signals simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Itaq universal syber green supermix, by Bio-Rad (1 mentions)
Cfx manager v 2, by Bio-Rad (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green pcr kit, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using cfx connect 96 real time pcr detection system
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantification of Target Bacteria in Digesta
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DNA was extracted from thawed digesta by a process involving repeated bead beating in the presence of high concentrations of SDS, salt and EDTA, followed by purification using QIAamp columns [25 (link)]. Purified DNA was quantified using a Thermo Scientific Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The numbers of target bacteria were estimated using quantitative real-time PCR using a CFX Connect 96 real-time PCR detection system and CFX Manager v 2.1 (Bio-Rad, CA, USA). Details of the primers and PCR reaction conditions for each of the targets have been described previously [26 (link)]. An 8-series of 10-fold dilutions of a sample-derived standard containing the target amplicon were analysed in parallel with DNA samples for estimation of absolute abundance and PCR efficiency.
3
RNA Extraction and Real-Time PCR Analysis
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After the fatty acid treatments, cells were directly lysed in the 25-cm2 flasks using RLT lysis buffer (Qiagen, Chadstone, Victoria, Australia), cell lysates were collected, and total RNA extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer instructions. cDNA was synthesised starting from 0.5 μg of total RNA as a template and using SuperScript II (Life Technologies Australia Pty. Ltd.) as described previously (40 (link)). Real-time PCR to assess the expression of IL-6 and ribosomal protein S18 was carried out using CFX Connect 96 real-time PCR detection system (Bio-Rad) employing a two-step cycling program of 95°C for 20 s, and then 40 cycles at 95°C for 3 s and 60°C for 30 s. Each reaction contained 1 μl of cDNA template, 5 μl of TaqMan fast advanced master mix, 3.5 μl of diethylpyrocarbonate-treated water, and 0.5 μl of either of the following Taqman assays: ribosomal protein S18 Hs01375212_g1 and IL-6: Hs00174131_m1. Ct values were normalised to the reference gene ribosomal protein S18 and data analysed using the comparative ΔCt method (41 (link)).
4
Quantifying HMGA2, MALAT1, and miR-145-5p
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The expression levels of HMGA2, MALAT1 and miR-145-5p in each cell line were detected by RT-qPCR. Total RNA was extracted from 1×106 cells using TRIzol® reagent according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a reverse transcriptase kit (cat. no. 639505; Takara Bio, Inc.). qPCR was performed with a CFX Connect 96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) using SYBR Green PCR kit (cat. no. KM4101; Kapa Biosystems; Roche Diagnostics). Each reaction was conducted in duplicate using the following thermocycling conditions: Denaturation at 95°C for 3 min, followed by 39 cycles of 95°C for 5 sec, 56°C for 10 sec and 72°C for 25 sec; 65°C for 5 sec and 95°C for 50 sec. The results were analyzed by the 2−ΔΔCq method (16 (link)). GAPDH and U6 were used as the reference genes for mRNA and miRNA, respectively. The primers were designed and provided by Wuhan Tianyihuiyuan Biotechnology Co., Ltd., and their sequences are listed in Table I .
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