For quantitative RT-PCR, total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA) or RNeasy Mini (Qiagen, Valencia, CA) and reverse transcribed by Superscript III (Invitrogen) or miRCURY LNA™ Universal RT microRNA PCR cDNA synthesis kit (EXIQON, Woburn, MA). cDNAs were used for PCR with SYBR Green reagents (Quanta Biosciences, Gaithersburg, MD) on MX3000 bioanalyzer (Stratagene, La Jolla, CA) and CFX Connect real-time PCR detection system (Bio Rad, Hercules, CA). The data was normalized to β-actin expression, or 5S rRNA expression for miR-203 expression. Product sizes were confirmed by gel electrophoresis. Primer sets for miR-203 and 5S rRNA were purchased from EXIQON. Additional primer sequences are provided in Table S1 .
Sybr green reagents
Manufactured by Quanta Biosciences
Sourced in United States
SYBR Green reagents are fluorescent dyes used in molecular biology applications, primarily for quantitative real-time PCR (qRT-PCR) analysis. They function by binding to double-stranded DNA, resulting in an increase in fluorescent signal that can be detected and quantified during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Viia7 real time pcr machine, by Thermo Fisher Scientific (2 mentions)
Mircury lna universal rt microrna pcr cdna synthesis kit, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Fluorescein diacetate, by Merck Group (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazole 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Perfecta sybr green fastmix reagent, by Quanta Biosciences (1 mentions)
Mc3t3 e1 preosteoblast cells, by American Type Culture Collection (1 mentions)
Qscript cdna synthesis kit, by Quanta Biosciences (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Sodium alginate, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Maxima probe rox qpcr master mix, by Thermo Fisher Scientific (1 mentions)
6 protocols using sybr green reagents
1
Quantitative RT-PCR Methodology
2
Gene Expression Analysis by qPCR
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Total RNA was isolated and purified using the RNeasy kit (Qiagen). cDNA was obtained by reverse transcribing 1-2 μg of total RNA using SuperscriptIII Reverse Transcriptase (Invitrogen) and used for qPCR. qPCR reactions were performed in triplicate using SYBR Green reagents (Quanta Biosciences) on a StepOnePlus (Life Technologies). GAPDH was used as an endogenous control. All results were normalized to GAPDH. Primers sets used are: GAPDH: 5’-AAGGTCGGAGTCAACGGATTT G-3’ and 5’-CCATGGGTGGAATCATATTGGAA-3’; SCCA1: 5’-AGCCGCGGTCTCGTGC-3’ and 5’-GGCAGCTGCAGCTTCTG-3’; SCCA2: 5’-AGCCACGGTCTCTCAG-3’ and 5-GCAGCTGCAGCTTCCA-3’; Serpinb3a: 5’-CATTTGTTTGCTGAAGCCACTAC-3’ and 5’-CATGTTCGAAATCCAGTGATTCC-3’; Serpinb3b: 5’-ATTCGTTTTCATGCAGCTGATGT-3’ and 5’-GAAAGCTGAAGTTAAATTTGTTCG-3’; PEA3: 5’-GGACTTCGCCTACGACTCA G-3’ and 5’-CGCAGAGGTTTCTCATAGCC-3’; IL-6: 5’-TCCACAAGCGCCTTCGGTCCA3’ and 5’-AGGGCTGAGATGCCGTCGAGGA-3’; IL-8: 5’-AAGGAAAACTGGGTGCAGAG-3’ and 5’-ATTGCATCTGGCAACCCTAC-3’; CXCL1: 5’-CACCCCAAGAACATCCAAAG-3’ and 5’-TAACTATGGGGGATGCAGGA-3’; G-CSF: 5’-ACTACAAGCAGCACTGCCCT-3’ and 5’-AGCAGTCAAAGGGGATGACA-3’; GM-CSF: 5’-CAAGTGAGGAAGATCCAGGG-3’ and 5’-AGAGAGTGTCCGAGCAGCAC-3’.
3
Fmoc-Phe-Phe-OH Hydrogel Cytotoxicity Analysis
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Lyophilized Fmoc-Phe-Phe-OH (FmocFF) was purchased from Bachem (Budendorf, Switzerland). Sodium alginate, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), fluorescein diacetate, propidium iodide, 4-Methylumbelliferyl phosphate (4-MUP), Rhodamin B, and Alizarin red were purchased from Sigma Aldrich (Rehovot, Israel). MC3T3-E1 preosteoblast cells were purchased from ATCC (Manassas, VA, USA). Pure Link RNA Mini Kit was purchased from Invitrogen; Thermo Fisher Scientific (Carlsbad, CA, USA), qScript cDNA Synthesis Kit and SYBR green reagents with Perfecta SYBR Green FastMix reagent with specific gene primers were purchased from Quanta Biosciences (Boston, BA, USA).
4
Gene Expression Analysis by qPCR
5
Quantifying Antimicrobial Peptide Expression
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Fresh ileum tissue was snap-frozen and stored at −80°C until RNA isolation. The RNA was extracted with TRIzol (Life Technologies), and cDNA synthesized at 37°C using a cDNA synthesis kit (Thermo Applied Biosystems). Gene expression of anti-microbial peptides (AMPs) and various cytokines were quantified using previously reported custom-made primer sets.16 (link) Real-time (RT)-PCR was performed using SYBR Green reagents (Quanta Biosciences, Beverly, MA, USA) on a ViiA 7 Real-time PCR machine (Thermo Fisher Scientific). Relative expression of these target genes compared to internal controls (HPRT or GAPDH) was calculated.
6
Quantification of Intestinal Antimicrobial Peptides and Cytokines
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Fresh ileal tissue (7 mm-long fragment) was snap frozen with liquid nitrogen and stored at −80 °C until RNA isolation. Total RNA was extracted with TRIzol (Life Technologies), and cDNA synthesized at 37 °C using a cDNA synthesis kit (Thermo, Applied Biosystems). Gene expression of anti-microbial peptides (AMPs) was assessed using primer sets: Reg3A (Mm00441128_g1), S100A8 (Mm00496696_g1), Tff3 (Rn00564851_m1), FFAR2/Gpr43 (Mm01176528_g1), and HPRT (Mm01545399_m1) as the housekeeping gene, using the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific, Grand Island, NY, USA). Gene expression of various cytokines was quantified using custom-made primer sets: IFNγ(forward: 5′-GCG TCA TTG AAT CAC ACC TG-3′, reverse: 5′-TGA GCT CAT TGA ATG CTT GG-3′)47 (link), IL-10 (forward: 5′-GGT TGC CAA GCC TTA TCG GA-3′, reverse: 5′-ACC TGC TCC ACT GCC TTG CT-3′)48 (link), IL-17A (forward: 5′-CTC AAA GCT CAG CGT GTC CAA ACA-3′, reverse: 5′-TAT CAG GGT CTT CAT TGC GGT GGA-3′) and GAPDH (forward: 5′-TCA ACA GCA ACT CCC ACT CTT CCA-3′, reverse: 5′-ACC CTG TTG CTG TAG CCG TAT TCA-3′)49 (link), as the housekeeping gene, using SYBR Green reagents (Quanta Biosciences, Beverly, MA, USA) performed on a ViiA 7 Real-time PCR machine (Thermo Fisher Scientific). Relative expression of these target genes compared to internal controls (HPRT or GAPDH) was calculated.
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