FACS purified cells were sorted directly into QIAZOL (Qiagen), vortexed and frozen immediately on dry ice. Total lung tissue was washed in PBS and placed directly in to RLT buffer (Qiagen). Gut tissue was opened longitudinally, scraped with a glass slide to remove mucus, and washed briefly in PBS then placed in RLT buffer + 10μM/ml β-mercaptoethanol (Sigma). Total RNA was isolated using the RNeasy micro kit or miRNeasy micro kit (Qiagen), according to the manufacturer’s instruction. mRNA was reverse transcribed to cDNA using random hexamers and the RevertAid First Strand cDNA synthesis kit (ThermoFisher). MicroRNAs were amplified and reverse transcribed using the Taqman advanced miRNA cDNA synthesis kit. Reactions for detection of Socs1, Gfi1, Il33, miR-142-3p, miR-142-5p, Gapdh, and snRNA U6 were carried out in 10 μl volumes using 2x Maxima probe/ROX qPCR master mix (Thermo Scientific) and Taqman real-time qPCR assays with FAM labelled probes were performed. Reactions for detection of Dclk1, Il25, and Gapdh were carried out in 10 μl volumes using fast SYBR-green mix (Applied Biosystems) master mix. Assays were analysed with an ABI Prism 7900HT real-time PCR instrument or ViiA 7 real-time PCR system (Applied Biosystems), where the Ct values were extracted. Relative gene expression and fold changes were calculated using the 2-ΔΔCt method.
Fast sybr green mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fast SYBR Green Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) that contains SYBR Green I dye, DNA polymerase, dNTPs, and optimized buffer components. The mix is designed to enable rapid and sensitive detection and quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Revertaid, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Mmlv reverse transcription system, by Thermo Fisher Scientific (3 mentions)
Quantstudio 5 384 well block real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna extraction kit, by Takara Bio (2 mentions)
Iscript reverse transcriptase supermix, by Thermo Fisher Scientific (2 mentions)
Trifast, by Avantor (2 mentions)
Stepone rt pcr 7 500 system, by Thermo Fisher Scientific (2 mentions)
Cfx384 touchtm real time pcr detection system, by Bio-Rad (2 mentions)
Rneasy microrna isolation kit, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
43 protocols using fast sybr green mix
1
Isolation and Quantification of RNA from Tissues
2
Quantitative RT-PCR Gene Expression Analysis
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RNA samples (500 ng) were reverse transcribed to obtain cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.S.A.). qPCR was performed using the FAST SYBR Green Mix (Applied Biosystems, U.S.A.), 0.3 μM of specific primers (Supplementary Table 3 ) and ~5 ng of cDNA. ΔΔCT-based relative quantification method was adopted for qPCR analysis using the QuantStudio 5 384-well Block Real-Time PCR system (Applied Biosystems, U.S.A.). The threshold cycle was determined to be 40. Data is presented as fold-change where CT values were normalised to β-ACTIN. Data presented are representative of three independent experiments with error bars indicative of the standard deviation (s.d.) unless otherwise stated.
3
Investigating Gmnn-Zic1 Interaction in HEK 293 Cells
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Total RNA was extracted using Clontech Nucleospin RNA extraction kit (#740955.250) using the manufacturer’s protocol. cDNA was synthesized using the Bio Rad iScript Reverse Transcriptase Supermix (#170-8841) followed by quantitative RT-PCR analysis using the Fast SYBR green mix from Applied Biosystems (#4385612) and gene-specific primer sequences shown in Supplementary Table S5 . Anti-Gmnn antibody from Santa Cruz (Cat. No. #sc-13015) and anti-Zic1 antibody from Rockland Inc. (Cat. #200-401-159) were used for immunoblotting at 1:200 dilution. The Gmnn and Zic1 bands were quantified using Bio-Rad Image Lab software. Co-immunoprecipitation experiments were performed as previously described16 (link) in HEK 293 cells transfected with HA-tagged Gmnn and Myc-tagged Zic1. Immunoprecipitation was performed with the HA tag using HA antibody (Abcam: ab9110) and blotted for Myc using Myc antibody (Abcam: ab32).
4
Quantification of Gene Expression by RT-qPCR
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Total RNA was isolated from the cells using TRIzol® reagent (Invitrogen, Tastrup, Denmark) according to manufacturer's protocol. After 21 days of differentiation, the samples were dissociated in TRIzol using the gentleMACS Dissociator (Miltenyi Biotec, Lund, Sweden). cDNA was synthetized from 1 μg of total RNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA). Primers were designed using Primer-BLAST software. Primer sequences are described in Table 1 (Supplementary Figures available here ). Real-time PCR was performed in the StepOnePlus Real-Time PCR System (Applied Biosystems) using Fast SYBR® Green Mix (Applied Biosystems, Foster City, CA, USA). Each sample was run in triplicates. The results were calculated using ΔCt method. The data are presented as 2−ΔΔCt, giving the relative expression change between day 0 and day 21.
5
RT-qPCR for Gene Expression Analysis in Mice
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Cells were harvested and rinsed in PBS prior to lysis in Buffer RLT (Qiagen) plus 10 μl/ml β-ME. RNA was extracted using an RNeasy MiniRNA isolation kit (QIAGEN) and complementary DNA libraries produced using RevertAid (Fisher) with random hexamer primers. RT-qPCR was completed using Fast SYBR Green Mix (Applied Biosystems) run on a CFX384 Touch real-time PCR detection system (Bio-Rad) or TaqMan Gene Expression Master Mix (Applied Biosystems) with fluorescein amidite probes. All kits were used following manufactures instructions. Samples were run in technical triplicate, with no template controls and melting curves included for quality control. ΔCt values were normalized to the housekeeping gene Hprt1. Primers were verified via BLAST against the Mus musculus genome via ensemble.org . Primer sequences are outlined in Supplementary Table 1 .
6
Real-Time qPCR Expression Analysis
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RNA was isolated from cells and tissues using the mRNeasy Kit (Qiagen) or Trifast (PeqLab) and reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Fermentas) according to manufacturer's protocols. Expression of mRNA was measured using FastSybrGreen Mix (Applied Biosystems) in a StepOne RT-PCR 7,500 system (Applied Biosystems). Ct-values of target genes were normalized to mHPRT and expressed as fold mean WT control using efficiencies calculated by LinReqPCR16 (link) for each amplification. The following primers were used:HPRT: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG; PTEN: for: ACACCGCCAAATTTAACTGC; rev: TACACCAGTCCGTCCCTTTC; IL-10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG; Arg1: for: GGAAAGCCAATGAAGAGCTG; rev: GCTTCCAACTGCCAGACTGT; Fizz: for: CTGGATTGGCAAGAAGTTCC; rev: CCCTTCTCATCTGCATCTCC; Stab1: for: CCCTCCTTCTGCTCTGTGTC; rev: CAAACTTGGTGTGGATGTCG; IL-23a: for: ATGCTGGATTGCAGAGCAGT; rev: ACGGGGCACATTATTTTTAG; Ym1: for: TTTCTCCAGTGTAGCCATCCTT; rev: TCTGGGTACAAGATCCCTGAA.
7
Quantitative RT-PCR Analysis of Colonic Immune Genes
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RNA was isolated from colon lamina propria single cell suspension using Trifast (PeqLab) and reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Fermentas) according to manufacturer’s protocols. Expression of mRNA was measured using FastSybrGreen Mix (Applied Biosystems) in a StepOne RT-PCR 7500 system (Applied Biosystems). Ct-values of target genes were normalized to mHPRT and expressed as fold mean WT control using efficiencies calculated using LinReqPCR (15 (link)) for each amplification. The following primers were used:
hprt: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG;
il10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG;
ifng: for: TGAGCTCATTGAATGCTTGG; rev: ACAGCAAGGCGAAAAAGGAT,
il17a; for: TGAGCTTCCCAGATCACAGA; rev: TCCAGAAGGCCCTCAGACTA,
foxp3: for: GCGAAAGTGGCAGAGAGGTA; rev: TCCAAGTCTCGTCTGAAGGC,
il6: for: CAAGTCGGAGGCTTAATTACACATG; rev: ATTGCCATTGCACAACTCTTTTCT.
hprt: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG;
il10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG;
ifng: for: TGAGCTCATTGAATGCTTGG; rev: ACAGCAAGGCGAAAAAGGAT,
il17a; for: TGAGCTTCCCAGATCACAGA; rev: TCCAGAAGGCCCTCAGACTA,
foxp3: for: GCGAAAGTGGCAGAGAGGTA; rev: TCCAAGTCTCGTCTGAAGGC,
il6: for: CAAGTCGGAGGCTTAATTACACATG; rev: ATTGCCATTGCACAACTCTTTTCT.
8
Quantifying MMP-9 mRNA Expression by qRT-PCR
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MMP-9 mRNA levels were measured by Real-Time PCR (7500 Fast system, Applied Biosystems, Carlsbad, CA, USA), using Fast Sybr Green Mix (Applied Biosystems). Results, normalized to actin beta, were calculated as fold change using the comparative threshold cycle method (2−ΔΔCT) relative to control cells (i.e., controls are assigned a value of 1 by definition). Primers (Hylabs, Israel) were designed (Primer-Express software, Applied-Biosystems) in different exons in order to eliminate DNA contamination.
MMP-9 forward primer: GCCACTACTGTGCCTTTGAGTC and MMP-9 reverse primer: CCCTCAGAGAATCGCCAGTACT.
Actin beta forward primer: CCTGGCACCCAGCACAAT and actin beta reverse primer: GCCGATCCACACGGAGTACT.
MMP-9 forward primer: GCCACTACTGTGCCTTTGAGTC and MMP-9 reverse primer: CCCTCAGAGAATCGCCAGTACT.
Actin beta forward primer: CCTGGCACCCAGCACAAT and actin beta reverse primer: GCCGATCCACACGGAGTACT.
9
RNA Isolation and qPCR Analysis
10
Quantitative Real-Time PCR Analysis
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RNA samples (250–500 ng) were reverse transcribed to obtain cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.S.A.). qPCR was performed using the FAST SYBR Green Mix (Applied Biosystems, U.S.A.), 0.3 μM of specific primers (Additional file 15 : Table S3) and ~ 5 ng of cDNA. ΔΔCT-based relative quantification method was adopted for qPCR analysis using the QuantStudio 5 384-well Block Real-Time PCR system (Applied Biosystems, U.S.A.). The threshold cycle was determined to be ≥ 35. Data are presented as fold-change where CT values were normalized to β-ACTIN. Data presented are representative of three independent experiments with error bars indicative of the standard deviation unless otherwise stated.
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