HT29-MTX E12 cells treated as above were frozen at −80°C with 1 ml RiboZol RNA extraction reagent (VWR, USA) and processed according to the manufacturer’s instructions. The Bio-Rad iScript Reverse Transcription Supermix for RT-qPCR was used to produce cDNA; it was performed with the SsoAdvanced Universal SYBR Green Supermix on a C1000 Touch CFX96 Real-Time System (Bio-Rad Laboratories, USA). Human patient cDNA was obtained from Origene Tissue scan TSC10765-ccrt502 (Origene, USA). Full details concerning these samples can be found at: ccrt502/tissuescan-crohnscolitis-cdna-array-ii">https://www.origene.com/catalog/tissues/tissuescan/ccrt502/tissuescan-crohnscolitis-cdna-array-ii ). Primer sequences can be found in Supplemental Table 1.
C1000 touch cfx96 real time system
Manufactured by Bio-Rad
Sourced in United States
The C1000 Touch CFX96 Real-Time System is a thermal cycler designed for real-time PCR applications. It features a 7-inch touchscreen interface, a 96-well sample block, and compatibility with various fluorescent dyes and probe chemistries.
Automatically generated - may contain errors
Lab products found in correlation
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Ribozol rna extraction reagent, by Avantor (2 mentions)
Tissue scan tsc10765 ccrt502, by OriGene (2 mentions)
Ccrt502, by OriGene (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Iscrip 8t, by Bio-Rad (2 mentions)
Mouse wg6 2.0 expression bead chip, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Absolute qpcr mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Quick rna miniprep, by Zymo Research (1 mentions)
Itaq universal sybr green chemistry, by Bio-Rad (1 mentions)
Onescript plus cdna synthesis kit, by Abmgood (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Itaq universal syber green supermix, by Bio-Rad (1 mentions)
Ssoadvanced universal probes supermix, by Bio-Rad (1 mentions)
14 protocols using c1000 touch cfx96 real time system
1
qRT-PCR Analysis of Crohn's and Colitis Samples
2
Quantifying mRNA Decay Kinetics
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Reverse transcription was performed using 100–200 ng of BrU-enriched RNA sample in a 20 µL reverse transcription reaction containing MultiScribe reverse transcriptase (Applied Biosystems). The reaction was incubated at 25°C for 10 min followed by an incubation at 37°C for 120 min and then at 85°C for 5 min. After reverse transcription, PCR reactions were performed using the C1000 touch CFX96 real time system (Bio-Rad) according to the manufacturer's protocol. Each 20 µL reaction contains 1× TaqMan Gene Expression Assay (Applied Biosystems), which has premixed TaqMan MGB probes and primers, 1× TaqMan Universal Master MixII (Applied Biosystems), which has DNA polymerase, dNTP, salt and buffer, and 2 µL of cDNA from reverse transcription. Half-lives of mRNAs were determined by least squares regression of each time point data set to a one-exponential decay equation (Chen et al. 2008 (link)).
3
Quantifying RNA Expression Changes
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RNA was isolated using the RNeasy mini kit (Qiagen). Reverse transcription was carried out using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was carried out using the ABsolute QPCR Mix, SYBR Green, no ROX (Thermo Fisher) on the C1000 Touch CFX96 Real-Time System (BioRad). In order to detect the right boundary of exon 1 and exon 2 after recombinase mediated correction of the genomic inversion, specific primers (47 and 48) in exon 1 and exon 3 were used for quantification. F8 mRNA expression was normalized to TATA-Box Binding Protein (TBP).
4
Profiling FSTL-1-dependent Gene Expression
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Total RNA was isolated from cells using the RNeasy kit (Qaigen), and cDNA was synthesized using iScrip 8t (Bio-Rad) according to the manufacturers instructions. Gene expression was analyzed by qPCR performed using primer/probe pairs for Hprt, Fstl1, Il6, Csf3, Il17rc (Applied Biosystems) in the C1000 Touch/CFX96 Real-Time System (Bio-Rad). RNA quality was verified using the 2100 Bioanalyzer (Agilent) before undergoing microarray analysis performed on ST2 cells using Mouse WG8.0 and BMSCs using Mouse WG6 2.0 Expression Bead Chip (Affymetrix) at the Genomics and Proteomics Core Laboratories, University of Pittsburgh. We defined differential expression as significant by setting threshold levels of ≥1.1 fold-change and p<0.05 by t-test with Bonferroni correction. Gene lists meeting these criteria were cross-analyzed for common FSTL-1 dependent differentially expressed genes.
5
qRT-PCR Analysis of Crohn's and Colitis Samples
6
Profiling FSTL-1-dependent Gene Expression
7
Quantitative ChIP-qPCR Analysis
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qPCRs were performed with 5µL ChIP-ed DNA against a 10x dilution series of input DNA using iQ™ SYBR Green Supermix (Biorad) together with primers (Table S2
8
RNA Isolation and qPCR Analysis
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Total RNA was isolated from NIH3T3 or primary HDFs using Quick-RNA Miniprep (Zymo Research). cDNA was synthesized using OneScript Plus cDNA Synthesis Kit (Abmgood). qPCR assay was performed by subjecting 100 ng of cDNA to iTaq Universal SYBR Green chemistry (Bio-Rad) in a C1000 Touch CFX96 Real time System (Bio-Rad). Expression data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or Actin as an internal control, and the relative expression was calculated using the ΔΔCt method. Primers are listed in Table 1 . Relative expression of mRNA is expressed as 2-delta Ct values as described previously and fold change in expression was calculated by comparing the 2-delta Ct values of the treated sample with that of untreated control. All samples were analyzed in triplicate.
9
Quantification of TE Gene Expression
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Total cellular RNA was extracted using the Qiagen RNeasy Mini Kit (Austin, TX), following the manufacturer's instructions with some modifications. TE tissue (Rep 1: n = 7, Rep 2: n = 5, Rep 3: n = 9) was lysed in RLT buffer using a 22-gauge needle and vortexed. After the first RW1 wash solution step, 80 μL of DNase solution was added directly to the membrane and incubated for 15 min at room temperature. Pure RNA was dissolved in nuclease-free water, and a spectrophotometer was used to determine RNA concentration for each sample. The RNA samples were stored at -80 • C. The RNA (190 ng) was reverse transcribed into cDNA via the BioRad iScript cDNA synthesis kit following the manufacturer's instructions. RT-PCR was then performed on TE cDNA (6 ng) in duplicate using BioRad iTaq Universal SYBER Green Supermix and BioRad C1000 Touch CFX96 Real Time System.
Expression of IFNT, PTGS2, TM4SF1, C3, and FGFR2 was measured using the primers in Table 2 and GAPDH used as a reference gene. All primers were diluted to a concentration of 10 μM. Each plate had negative controls to assure no background contamination. The PCR program was 5 min at 95 • C for melting, 15 s at the given annealing temperature (Table 2) and 15 s at 70 • C for extension, for 40 cycles. All CVs were less than 20%. Amplicons were electrophoresed and were verified for identity by sequencing (Iowa State Genomics Core).
Expression of IFNT, PTGS2, TM4SF1, C3, and FGFR2 was measured using the primers in Table 2 and GAPDH used as a reference gene. All primers were diluted to a concentration of 10 μM. Each plate had negative controls to assure no background contamination. The PCR program was 5 min at 95 • C for melting, 15 s at the given annealing temperature (Table 2) and 15 s at 70 • C for extension, for 40 cycles. All CVs were less than 20%. Amplicons were electrophoresed and were verified for identity by sequencing (Iowa State Genomics Core).
10
Sclerotinia spp. Detection by Real-Time PCR
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Presence of Sclerotinia spp. in the samples was tested by a Taqman-real-time assay, essentially as described by Almquist and Wallenhammar (2015) . The reaction mixture consisted of SsoAdvanced Universal Probes Supermix (Biorad), 0.2 μM of each primer, 0.2 μM of TaqMan probe and 2 μL of DNA in a 25 μl reaction volume. The real-time PCR was run on a C1000 Touch™ CFX96™ Real-Time System (Biorad) with cycling conditions as described by Almquist and Wallenhammar (2015) . Three replicates of each DNA sample were tested. If at least two replicates of three were amplified by the PCR protocol, the sample was considered positive for Sclerotinia spp. Reference isolates of S. sclerotiorum and S. subarctica sequenced in 2013 at the University of Warwick, England, were used as positive controls (Brodal et al. 2017 ).
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