For 263 patients whose DNA was available, rs8099917 genotyping was performed by PCR amplification with ABI PRISM ® 7000 SDS (Applied Biosystems, Foster City, CA, USA). The PCR amplification mixture was 10 µL in a total volume containing 1.5 µL DNA template, 5 µL 2 × TaqMan Genotyping Master Mix, 0.25 µL 40 × TaqMan SNP Genotyping Assay Mix (rs8099917, C_11710096_10), and 3.25 µL ultrapure water. The PCR reactions were performed in 96-well microplates with three negative control (NTC). Reaction conditions were: 50 cycles of thermal denaturation at 95 ℃ for 5 minutes, denaturation at 92ºC for 20 seconds and annealing at 60ºC for 1 minute. Two allelic-specific TaqMan probes were used to detect a specific SNP target. Allele discrimination was achieved by the detection of fluorescent signal and analysis of the distribution of the scatter plot chart using ABI PRISM ® 7000 SDS (Applied Biosystems, Foster City, CA, USA).
Abi prism 7000 sds
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
The ABI Prism 7000 SDS is a real-time PCR instrument designed for quantitative gene expression analysis. It utilizes the 7000 Sequence Detection System technology to perform real-time monitoring of amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Rnase free dnase, by Roche (3 mentions)
Sybr green real time pcr kit, by Thermo Fisher Scientific (3 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Abi steponeplus system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sybr green system, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Qiaquick spin column, by Qiagen (1 mentions)
Rnalater, by Qiagen (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Rneasy spin column, by Qiagen (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
High capacity reverse transcription kit, by Promega (1 mentions)
24 protocols using abi prism 7000 sds
1
Genotyping of rs8099917 SNP by TaqMan Assay
2
Quantitative Gene Expression Analysis
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According to standard procedures, cDNA was synthesized with Superscript III (Invitrogen-Life Technologies Italia, Monza, Italy) and mRNA expression was analyzed using the SYBR-Green qRT-PCR method (5 ng/assay) (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Quantification was achieved with ABI Prism 7000 SDS (Applied Biosystems, Monza, Italy). mRNA expression value was then normalized for beta 2-microglobulin levels.
Table2 reports the primers used to this aim. Micro-RNA (miRNA) levels were analyzed using the Applied Biosystems TaqMan quantitative qRT-PCR method (1 ng/assay) performed according to the manufacturer's instructions and quantified with the ABI Prism 7000 SDS (Applied Biosystems, Monza, Italy). Mature miRNA levels were normalized to miR-16, whose expression is constant in all tested RNA samples. For both mRNAs and miRNAs, relative expression was calculated using the comparative Ct method (2-ΔΔCt).
Table
3
Quantitative Gene Expression Analysis
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Total RNA was extracted from the tendon tissues of the mice using TRIzol® Reagent (Ambion™) (Applied Biosystems, MA, USA), and 500 ng of RNA was reverse-transcribed into cDNA using TaqMan® Reverse Transcription Reagents (Thermo Fisher Scientific, MA, USA). The expression of target genes was examined using ABI Prism SDS 7000 (Thermo Fisher Scientific) at 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min, and then calculated using the 2−ΔΔCt method. Primer sequences for qRT-PCR are listed in Table S1 . GAPDH was used as an internal control.
4
Quantitative RT-PCR for Immune Genes
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Total RNA was extracted using Trizol reagent (Invitrogen Canada, Burlington, Canada) according to the manufacturer’s instructions. SuperScript II reverse transcriptase (Invitrogen Canada) was used to reverse transcribe the RNA and the PCR reaction was performed using the SYBR® green system (Applied Biosystems, Foster City, CA, USA). Reactions were monitored on an ABI Prism SDS 7000 (Thermo Scientific, Waltham, MA, USA) machine and results were analyzed with SDS 2.0 software.
The housekeeping gene HPRT (hypoxanthine-guanine phosphoribosyl transferase) was used as an internal control. Primers (Sigma-Aldrich) used were as follows: HPRT (accession number: NM_ J00423): left: 5′-caagcttgctggtgaaaagga-3′, right: 5′-tgaagtactcattatagtcaagggcatatc-3′; IL-1β (accession number: NM_ M15131): left: 5′-gtggaacttgaggccacatt-3′, right: 5′-tgtgacaaaaatgcctggaa-3′; iNOS (accession number: NM_ BC062378): left: 5′-caccttggagttcacccagt-3′, right: 5′-accactcgtacttgggatgc-3′, and IL-6 (accession number: NM_ M24221): left: 5′-ccggagaggagacttcacag-3′, right: 5′-tccacgatttcccagagaac-3′, with the following primer cycling conditions: 95 °C for 15 s, 58 °C for 50 s, and 72 °C for 15 s (40 cycles).
The housekeeping gene HPRT (hypoxanthine-guanine phosphoribosyl transferase) was used as an internal control. Primers (Sigma-Aldrich) used were as follows: HPRT (accession number: NM_ J00423): left: 5′-caagcttgctggtgaaaagga-3′, right: 5′-tgaagtactcattatagtcaagggcatatc-3′; IL-1β (accession number: NM_ M15131): left: 5′-gtggaacttgaggccacatt-3′, right: 5′-tgtgacaaaaatgcctggaa-3′; iNOS (accession number: NM_ BC062378): left: 5′-caccttggagttcacccagt-3′, right: 5′-accactcgtacttgggatgc-3′, and IL-6 (accession number: NM_ M24221): left: 5′-ccggagaggagacttcacag-3′, right: 5′-tccacgatttcccagagaac-3′, with the following primer cycling conditions: 95 °C for 15 s, 58 °C for 50 s, and 72 °C for 15 s (40 cycles).
5
Quantitative RT-PCR of EMT Markers
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Using TRIzol® Reagent (Ambion™) and TaqMan® Reverse Transcription Reagents (Applied Biosystems, Inc.; Thermo Fisher Scientific, Inc.), total RNA was extracted, and complementary DNA (cDNA) was reverse-transcribed. Each well (25 μL PCR reaction volume) included 12.5 μL of SYBR Green Mix (Power SYBR® Green PCR Master Mix, Applied Biosystems, Inc.), 0.2 μL of cDNA, 1 μL of primer pair mix (5 pmol/μL each primer) and 11.3 μL of DNAse/RNAse-free H2O. Each sample was prepared in three replicates. PCR was performed using ABI Prism SDS 7000 (Applied Biosystems, Inc.; Thermo Fisher Scientific, Inc.). Specific primers were synthesized by Takara Biotechnology Co., Ltd. The sequences of primers were as follows: HMGB1, forward: 5′-GCCTCCTTCGGCCTTCTT-3′ and reverse: 5′-ACAGGCCAGGATGTTCTCCTTT-3′; E-cadherin, forward: 5′-GGATTGCAAATTCCTGCCATTC-3′ and reverse: 5′-AACGTTGTCCCGGGTGTCA-3′; N-cadherin, forward: 5′-GTAGCTAATCTAACTGTGACCGATAAGG-3′ and reverse: 5′-TTGGTTTGACCACGGTGACTAA-3′; vimentin, forward: 5′-GCAGGAGGCAGAAGAATGGTA-3′ and reverse: 5′-GGGACTCATTGGTTCCTTTAAGG-3′; Snail, forward: 5′-TCGGAAGCCTAACTACAGCGA-3′ and reverse: 5′-AGATGAGCATTGGCAGCGAG-3′; Twist, forward: 5′-AGCAAGATTCAGACCCTCAAGCT-3′ and reverse: 5′-CCTGGTAGAGGAAGTCGATGTACCT-3′; β-actin, forward: 5′-CCACGAAACTACCTTCAACTCCA-3′ and reverse: 5′-GTGATCTCCTTCTGCATCCTGTC-3′.
6
RT-qPCR Gene Expression Analysis
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Prior to cDNA preparation, all samples were stored in RNAlater (Qiagen, Hilden, Germany) at −20°C. Where tissues were processed, samples were disrupted using a TissueLyser LT (Qiagen). RNA was extracted from samples using RNeasy spin columns (Qiagen) and extracted nucleic acids were subjected to DNaseI (Qiagen) treatment in solution and a further column cleanup. RNA for qRT-PCR was reverse transcribed using the Applied Biosystems (Carlsbad, CA, USA) high capacity reverse transcription kit with an added RNase-inhibitor (Promega Biosciences, Madison, WI, USA) and cDNA was cleaned using QIAquick spin columns (Qiagen). All elutions were conducted with nuclease-free water (Qiagen).
Purified cDNA was used as template for the amplification of target gene transcripts with SYBR Green PCR master mix (Applied Biosystems) using the ABI Prism SDS 7000 and 7900HT machines (Applied Biosystems). Target gene expression was determined relative to Hprt using the ΔCT method using previously-described primer sets and methodology [21 (link)]. In plots showing expression, a hashed line indicating the theoretical detection limit is shown. Fold change values are calculated against an unstimulated control, represented by the hashed line, which is standardized to 1.
Purified cDNA was used as template for the amplification of target gene transcripts with SYBR Green PCR master mix (Applied Biosystems) using the ABI Prism SDS 7000 and 7900HT machines (Applied Biosystems). Target gene expression was determined relative to Hprt using the ΔCT method using previously-described primer sets and methodology [21 (link)]. In plots showing expression, a hashed line indicating the theoretical detection limit is shown. Fold change values are calculated against an unstimulated control, represented by the hashed line, which is standardized to 1.
7
Quantifying Gene Expression in Tissues
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Total RNA was extracted from lung or skin with TRIzol (Invitrogen) and further purified with RNAeasy columns (Qiagen, Carlsbad, CA) and in column DNase digestion (Qiagen). One microgram of total RNA was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out in triplicate with a 10–20-fold dilution of first-strand cDNA using taqman probes and primers purchased as Assays On Demand (Applied Biosystems, Foster City, CA) for the following genes: β-glucuronidase (GusB—reference gene control), HsHoxA5, MmTSP-2, MmEphrinA1, MmVEGF-A, MmCCL-2 and MmCXCL12. An ABI Prism SDS 7000 (Applied Biosystems) was used according to the manufacturer’s instructions with the following cycling protocol: one cycle of 50°C, 2 minutes; 95°C, 10 minutes; 40 cycles of 95°C, 15 seconds; 50°C, 1 minute. Data was analyzed with ABI Prism SDS 7000 companion software and Microsoft Excel.
8
Quantification of Immune-related Gene Expression in Keratinocytes
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Total RNA from keratinocyte cultures was extracted using the TRIzol reagent (Invitrogen); mRNA was reverse-transcribed into cDNA and analysed by real-time PCR. The expression of human SOCS3, S100A7, IL-20, HBD-2, LL-37, and HPRT-1 mRNA was evaluated in the ABI Prism SDS 7000 PCR instrument (Applied Biosystems, Branchburg, NJ), using SYBR Green PCR reagents or TaqMan PCR Master Mix. The same PCR tools were employed to analyse murine IL-17A, IL-22, IFN-γ, TNF-α, CXCL10, CCL2, CCL20, CXCL16, and IL-6 mRNAs. The forward and reverse primers employed for real-time PCR for SOCS3 were 5′-AAGGACGGAGACTTCGATTCG-3′ and 5′-AAACTTGCTGTGGGTGACCAT-3′, and for LL-37 5′-TTTTGCGGAATCTTGTACCCA-3′ and 5′-TCTCAGAGCCCAGAAGCCTG-3′. The sequences of the primers for β-defensin- (HBD-) 2 mRNA have been previously described [26 (link)]. Primers for S100A7, IL-20, and HPRT-1 were provided by Applied Biosystems (HS 00161488, HS 00218888, and HS 4333768, respectively). Primers used for the detection of murine molecules were retrieved from previous studies [27 (link)]. Human and murine mRNA level values were normalized to HPRT-1 and β-2-microglobulin mRNA, respectively. The values obtained from triplicate experiments were averaged, and data presented as mean 2−ΔΔCT ± SD.
9
Quantitative PCR Gene Expression Analysis
10
Murine Intestine RNA Expression
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Total RNA was extracted from murine small intestines and HepG2 cells using TRIzol. mRNA expression was measured using RT-PCR, as described previously (20, 21) . The oligonucleotide sequences for each mRNA target are shown in Table 1. Data were analyzed using ABI Prism 7000 SDS software (Life Technologies), using the multiplex comparative method.
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