Total RNA was isolated using RNeasy Plus mini kit (74104; Qiagen, Valencia, CA) and reverse transcribed using Superscript III First‐Strand Synthesis Supermix (18080400; Invitrogen, Carlsbad, CA). Quantitative real‐time polymerase chain reaction was performed using the SYBR green detection kit (4385612; Invitrogen). Expression values were normalized with glyceraldehyde 3‐phosphate dehydrogenase before calculating expression ratios. Primer sequences used in expression analysis are available upon request.
Sybr green detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SYBR Green detection kit is a reagent designed for real-time PCR analysis. It contains the SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon excitation. This allows for the detection and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Wisent (1 mentions)
L leu, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Wisent (1 mentions)
Hmb free acid, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Trypsin, by Wisent (1 mentions)
Cfx real time pcr detection system, by Bio-Rad (1 mentions)
7900 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miscript 2 kit, by Qiagen (1 mentions)
Qiaamp dna stool kit, by Qiagen (1 mentions)
Pmdtm19 t vector cloning kit, by Takara Bio (1 mentions)
7 protocols using sybr green detection kit
1
RNA Extraction and RT-qPCR Analysis
2
Molecular Profiling of Muscle Cell Differentiation
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L-Leu (purity ≥ 98.5-101.0%), KIC (purity ≥ 98 %), and HMB free acid (purity ≥ 95%) were purchased from Sigma (St. Louis, MO, USA). TRIzol, DNase I, and SYBR Green detection kit were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). Protease inhibitor cocktail was purchased from Roche (Basel, Switzerland). Phosphatase inhibitors were purchased from Thermo Scientific (Waltham, MA, USA). Phosphate Buffered Saline (PBS) and Trypsin were also purchased from Wisent. Mesotrione (2-(4-Mesyl-2-nitrobenzoyl)-1,3-cyclohexanedione)-Pestanal©, catalogue No. 33855) was obtained from Fluka (St. Louis, MO, USA). The growth medium used for cell growth consisted of high glucose Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (FBS) (Gibco #26050-088), and 1% Antibiotic-Antimycotic (Wisent #450-115-EL). The medium used for differentiation of cells was high glucose DMEM supplemented with 2% horse serum (HS) (Gibco #26050088) and 1% Antibiotic-Antimycotic.
3
RNA Extraction and qRT-PCR Analysis
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RNA of the OVCAR8 or NCI_ADR/RES cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) following the product manual. The concentration of RNA was determined by measuringthe absorbance at 260nm in Nanodrop(Thermo Fisher Scientific), and the quality was examined by the ratio of absorbance at 260-280 nm. Then RNA was reverse transcribed into cDNA with Promega A5000 Reverse Transcription System (Promega, Madison, WI) according to the manufacturer's protocol. Quantitative real-time RT-PCR analysis was performed on Bio-Rad CFX real-time PCR detection system (Bio-rad) using SYBR Green detection kit (Applied Biosystems) according to the manufacturer's instructions. The specific primers for MDR1 were 5'-GGGAGCTTAACACCCGACTTA-3' (sense) and 5'-GCCAAAATCACAAGGGTTAGCTT-3'(antisense), for Sirt4 were 5'-CCGTAGAGCTGTGAGAGAATGAA-3'(sense) and 5'-AGGGTCCAGAGGAGGACTTG -3'(antisense), for PCCA were 5'-AAGCTACCTCAACATGGATGC-3' (sense) and 5'-GTGTCAGGTCCAATGAAAACGA-3'(antisense), for Foxred1 were 5'-CTCAGTAGGTGGGATTTGTCAGC-3' (sense) and 5'-CTCTGCACTTTCACGTTGCTC-3' (antisense), and for GAPDH were 5'-CTTCACCACCATGGAGGAGGC-3' (sense) and 5'-GGCATGGACTGTGGTCATGAG-3' (antisense). The relative quantification of gene expression was analyzed by the 2-ΔΔCT method. Real-time quantitative RT-PCR analysis was repeated at least three times.
4
Gene Expression Analysis by RT-qPCR and Western Blot
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Total RNA was extracted with TRIzol reagent and reverse transcribed into cDNA according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Quantitative real-time PCR analysis was performed using TheABI StepOne PLUS and SYBR Green detection kit according to the manufacturer’s instructions (Applied Biosystems).
For Western blotting, the indicated proteins were separated by SDS-PAGE and electro-transferred to PVDF membranes, which were blocked with 5% non-fat milk and then probed with primary antibodies. The protein bands were visualized with enhanced chemiluminescence substrate after probing with the indicated secondary antibodies. GADPH was used as loading control.
For Western blotting, the indicated proteins were separated by SDS-PAGE and electro-transferred to PVDF membranes, which were blocked with 5% non-fat milk and then probed with primary antibodies. The protein bands were visualized with enhanced chemiluminescence substrate after probing with the indicated secondary antibodies. GADPH was used as loading control.
5
Quantifying Gene Expression via RT-PCR
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Gene expression was measured by real-time polymerase chain reaction (RT-PCR), as previously described (25 (link)). Briefly, total RNA was isolated from colonic tissues using TRIzol (Invitrogen, Carlsbad, CA, USA), and fluoresce were monitored by the SYBR Green detection kit (Thermo Fisher Scientific, Waltham, MA, USA) on a 7900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The RT-PCR was conducted with primers of the target genes (Supplementary Table 2 ) and the reference gene β-actin, and relative gene expression was calculated by the 2−ΔΔCt method (26 (link)).
6
RNA Isolation and qPCR Analysis
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RNA was isolated using TRIzol reagent (Thermo Fisher Scientific), and cDNA was synthesized using the miScript II kit (QIAGEN). Target cDNAs were detected using a SYBR green detection kit (Thermo Fisher Scientific) and gene-specific primers.
7
Quantifying Intestinal Microbial Abundances
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The abundances of the ileal and colonic microbiota were analyzed as previously described (Su et al., 2018 (link)). Briefly, the total microbial DNA was extracted and purified by a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany). The specific primers (Supplementary Table 2 ) of targeted microbiota were used to amplify the targeted gene fragments. The recombinant plasmid vector of targeted microbiota was constructed and cloned according to the pMDTM19-T vector cloning kit (TaKaRa Biotechnology, Dalian, China) instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed using an SYBR Green detection kit (Thermo Fisher Scientific, Waltham, MA) to determine the abundances of general microbial DNA from the intestinal contents and above recombinant DNA using a Lightcycler 480II instrument (Applied Biosystems; Su et al., 2018 (link)). The standard curves for all determined microbiota were constructed based on the recombinant DNA of representative species. The results are presented as the gene copies of microbial DNA of the intestinal contents.
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