Genotyping of rs671 by HRM was performed in 384-well plates on a 384-well 7900HT Fast Real-Time PCR System (7900 HT, Applied Biosystems, Foster City, CA) as previously described [9 (link)]. Briefly, two primers (as shown in Table 1 ) were designed to amplify a 78 bp fragment cover target locus rs671 and SYTO 9 (Invitrogen, Eugene, Oregon, USA), a saturating dye was added to the standard PCR reaction mixture before amplification. The PCR reaction mixture contained 5 μl 2x GC Buffer, 6.25 μmol of each dNTP, 0.25 U Taq DNA Polymerase (TaKaRa Biotechnology, Tokyo, Japan), 1 μl genomic DNA, 100 pmol SYTO 9 and 5 pmol of each primer in a reaction volume of 10 μl. PCR conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing/extending at 60°C for 1 min. After PCR, the samples were melted at a ramp rate of 1% from 60°C to 95°C. The fluorescence signals were measured during this process and the melting curve was automatically converted to melting peaks. SDS Software v2.3 and High Resolution Melt v2.0 (Applied Biosystems, Foster City, CA) were used for data analysis.
Sds software v2
Manufactured by Thermo Fisher Scientific
Sourced in United States
SDS Software v2.3 is a data management software suite developed by Thermo Fisher Scientific. The software's core function is to facilitate the storage, organization, and retrieval of safety data sheets (SDS) for chemical substances and mixtures. The software provides a centralized platform for managing SDS information, enabling users to access critical safety data quickly and efficiently.
Automatically generated - may contain errors
Lab products found in correlation
7900ht fast real time pcr system, by Thermo Fisher Scientific (8 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (4 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (3 mentions)
7900ht system, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Abi 7900ht instrument, by Thermo Fisher Scientific (2 mentions)
Quantifast sybr green pcr kit, by Qiagen (2 mentions)
7900ht fast rt pcr system, by Thermo Fisher Scientific (2 mentions)
Rq manager v1, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Takara Bio (1 mentions)
Syto 9, by Takara Bio (1 mentions)
Applied biosystems 7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
41 protocols using sds software v2
1
Genotyping of rs671 Variant by High-Resolution Melting
2
Quantitative miRNA Expression Analysis in PDAC
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Quantitative Real-Time (qRT) PCR was performed using the miScript PCR system (Qiagen). Total RNA samples (1μg) of PDAC cell lines were reverse transcribed to cDNA using miScript Reverse Transcription Kits (Qiagen). For each sample, 5μL of the generated cDNA was mixed with 10μL 2x QuantiTect SYBR, 2μL 10x miScript Universal Primer, 2μL gene specific 10x miScript Primer Assay and 1μL nuclease free water. All samples were analyzed in triplicate reactions using Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) with miScript SYBR Green PCR Kit (Qiagen). The cycling program involved preliminary denaturation at 95°C for 10min, followed by 40 cycles of denaturation at 95°C for 15s, annealing at 60°C for 60 s and elongation at 60°C for 60s. Quantitative miR analysis was performed using SDS-Software v2.3 (Applied Biosystems). Expression of each miR was analyzed quantitatively relative to the housekeeping genes RNU6-2, SNORD68, and SNORD96A by the 2–ΔΔCT method.
3
RNA Extraction and qPCR Analysis of FoxM1 Expression
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Total RNA from tissues or cells was extracted using TRIzol reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Complementary (c) DNA was produced with reverse transcription using the PrimeScript First Strand cDNA Synthesis kit (cat. no. DRR047A; Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR assays were performed using the MX3000P system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) and the SYBR Premix ExTaq kit (cat. no. DRR042A; Takara Biotechnology Co., Ltd.). GAPDH was used as an internal control. The primers for qPCR were as follows: FoxM1-Forward, GGA GCA GCG ACA GGT TAA GG; FoxM1-Reverse, GTT GAT GGC GAA TTG TAT CAT GG; GAPDH-Forward, TGA AGG TCG GAG TCA ACG G; GAPDH-Reverse, CTG GAA GAT GGT GAT GGG ATT. The reaction mixture consisted of 2 µl of cDNA template in a final reaction volume of 20 µl; there were three replicates of each sample. Initial denaturation at 95°C for 5 min was followed by 40 cycles of amplification at 95°C for 10 sec, 58°C for 20 sec and 72°C for 20 sec. A melting curve analysis was performed following the PCR cycles. Data was analyzed with SDS software v2.3 (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative expression levels were calculated using the 2−∆ΔCt method (24 (link)).
4
Quantitative Analysis of miRNA Levels
5
Analyzing miRNA Expression Profiles
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Data are presented as the mean values ± SD of individual experiments. Because of the nonparametric distribution of data, the Mann-Whitney U test was performed to assess statistical differences between the two experimental groups. In all cases a P value < 0.05 was considered significant. The miRNA gene expression values were normalized according to endogenous control RNU6B and relative expression values were obtained using the ΔCt method with SDS software v 2.3 (Applied Biosystems, CA, USA). The relative target miRNA expression levels were determined by the equation 2−ΔCt, in which ΔCt was calculated as follows: ΔCt = CtmiR-of-interest − Ct RNU6B. Different miRNA expression between the groups was identified according to fold change. Fold change cut-off was 2-fold, if the changes were observed in both cell samples simultaneously. The miRNA expression levels were visualized through heat maps (TIGR MultiExperiment Viewer 4.0, The Institute for Genomic Research, USA), dendrograms, and column charts. The Spearman correlation test was used to correlate miRNA expression data with interleukin production.
6
Genotyping of COL12A1 rs970547
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Genotyping for COL12A1 rs970547 polymorphism was performed in duplicate using an allelic discrimination assay on a 7900HT Fast real-time polymerase chain reaction instrument (Applied biosystems™, Life Technologies, 2012, Carlsbad, CA, USA) with TaqMan probes (TaqMan® Pre-Designed SNP Genotyping Assay ID: C_7580617_10). Genotypes were assigned using software (SDS Software v2.3, Applied BiosystemsTM). To ensure proper internal control, positive and negative controls from different DNA aliquots were used for each genotype analysis. The average genotyping rate was ~98%.
7
Quantitative PCR Gene Expression Analysis
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TaqMan PCR assays were used to measure RREB1 (Hs00366111_m1), INS (Hs00355773_m1), CAMK2A (Hs00392405_m1), GPR56 (Hs00173754_m1), RFX2 (Hs00172177_m1), RFX3, (Hs01060440_m1) and TBP (Hs00427620_m1) gene expression (ThermoFisher Scientific, UK). qPCR reactions were performed on a 7900HT Fast Real-Time PCR System (ThermoFisher Scientific, UK) using SDS software (v2.3; Applied Biosystems) and the following conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Cycle thresholds were transformed to gene copy numbers and normalised to the geometric mean of the housekeeping genes TBP and PPIA, except for Fig. 7 , in which gene expression was normalised to TBP only.
8
Gene Expression Analysis by qRT-PCR
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Cycle threshold (Ct) values were calculated using the SDS software v.2.3 (Applied Biosystems) using automatic baseline settings and a threshold of 0.2. GAPDH was used as endogenous control. Data are presented as target gene expression = 2−ΔCt, with ΔCt = (target gene Ct-GAPDH Ct). Gene expression was computed by real-time Statminer® software v.4.2 (Intergromics, Inc), using the Benjamini-Hochberg algorithm [35 ] with the FDR set at a value of 5%. Gene expression had to be detected in at least 50% of samples in each study group in order to be considered for analysis. PCR GEO accession number GSE52513.
9
RNA Isolation and qRT-PCR Analysis of Mouse Myocardial Tissue
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For RNA isolation of snap-frozen mouse myocardial tissue or NRVM, RNeasy Mini Kit (Qiagen) was used according to the manufacturer protocol. Total RNA (500 ng) was transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen) and qRT-PCR was performed in triplicates (20 ng cDNA per reaction) with appropriate primers in 5′–3′orientation (Table 1 ) using QuantiFAST SYBR Green PCR Kit including ROX dye (Qiagen). Forty-five cycles (95°C, 10 s; 60°C, 30 s) were performed on an ABI prism 7900HT Fast system (Applied Biosystems). Relative gene expression values, adjusted for the same CT-threshold and baseline settings, were calculated according to the 2−ΔΔCT method (40 (link)) using Gapdh as housekeeping gene (SDS Software v2.3, Applied Biosystems).
10
Profiling Kidney miRNA Signatures in DOCA-Salt Mouse Model
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To assess the different signature of the miRNAs of the mouse kidney induced by DOCA-salt treatment and the control, we profiled the production of 588 miRNAs in the kidney with the ABI TaqMan MicroRNA Array kit (Applied Biosystems) according to the manufacturer’s protocol. In brief, 333 ng of mouse kidney total RNA was reverse-transcribed with megaplex RT primers (Megaplex RT Rodent Pool A or B) followed by a pre-amplification reaction with megaplex preamp primers; subsequently, a real-time PCR with TaqMan Rodent MicroRNA Array (Pool A or B) was performed on an Applied Biosystems 7900HT System. SDS software V2.3, and RQ Manage1.2 (Applied Biosystems) were used to obtain the comparative threshold cycle (Ct) value. U6 small nuclear RNA included in the TaqMan Rodent MicroRNA Array was used as an endogenous control.
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