Total RNA was harvested from cell lines using the Rneasy Mini Kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized from 1 mg of total RNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Gene expression was measured by qPCR. TaqMan primers and probes (Life Technologies) were used for the following mouse genes: Tfap2c Mm00493473_m1, Egfr Mm00433023_m1 and Gapdh Mm00484668_m1. TaqMan primers and probes (Life Technologies) were used for the following human genes: TFAP2C Hs00231476_m1, EGFR Hs01076078_m1 and GAPDH Hs02758991_g1. Expression values were normalized to average Gapdh, the endogenous control.
Taqman primers and probe
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan
TaqMan primers and probes are oligonucleotide sequences designed for use in real-time PCR assays. They are used to detect and quantify specific DNA or RNA targets in samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (22 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (8 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (8 mentions)
Mirneasy mini kit, by Qiagen (7 mentions)
Abi prism 7300, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Nanodrop, by Thermo Fisher Scientific (5 mentions)
Dnase, by Promega (5 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (5 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (5 mentions)
Total rna isolation kit, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (4 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (4 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Abi prism 7300 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Rneasy micro kit, by Qiagen (4 mentions)
178 protocols using taqman primers and probe
1
Gene Expression Analysis of Tfap2c, Egfr, and Gapdh
2
Quantifying Interferon Response Genes by qRT-PCR
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Total RNA was extracted from tissues by homogenization in 0.5 ml ISOGEN (NipponGene) using a TissueLyser (Qiagen). The relative mRNA levels were quantified using the comparative C T method (∆∆C T method) by qRT-PCR using the TaqMan ® system. Hydroxymethylbilane synthase (HMBS) was used as an internal control. PCR amplification was performed in a 10-µl final reaction mixture containing 5 µl 2X Quantitect RT-PCR Master Mix, 0.1 µl Quantitect RT mix (Qiagen), 0.5 µl TaqMan probe and primers (Life Technologies), and 100 ng total RNA. Thermal cycling conditions comprised an initial step at 95˚C for 10 min, followed by 45 cycles at 95˚C for 10 sec, 60˚C for 10 sec and 72˚C for 5 sec. The TaqMan ® probe and primers for interferon response genes, interferon stimulated gene factor 3γ (ISGF-3γ), 2', 5'-oligoadenylate synthetase 2 (OAS2), interferon-induced myxovirus resistance protein 1 (MX1) and HMBS were purchased from Life Technologies. The 5'-fluorescent reporter dye fluorescence was detected using a LightCycler (Roche Diagnostics).
Statistical analysis. All in vitro experiments were performed in triplicate and repeated 3 times. Student's t-test was used to determine the significance of differences between the groups, with values of P<0.05 considered statistically significant.
Statistical analysis. All in vitro experiments were performed in triplicate and repeated 3 times. Student's t-test was used to determine the significance of differences between the groups, with values of P<0.05 considered statistically significant.
3
Skin Gene Expression Analysis
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At indicated times after tape stripping or S. aureus infection, total skin and epidermal and dermal sheet RNA was extracted with Total RNA Isolation Kit (Ambion). cDNA was prepared with iScript cDNA Synthesis Kit (Bio-Rad). PCR reactions were run on an ABI Prism 7300 (Applied Biosystems) sequence detection system platform. TaqMan primers and probes were obtained from Life Technologies. The housekeeping gene β2-microglobulin was used as an internal control.
4
Quantifying Placental Gene Expression
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Total RNA was isolated from PDMSCs-CM-treated and untreated PE placentae using TRIzol reagent (Life Technologies, Invitrogen, Monza, Italy) according to manufacturer instructions. Genomic DNA contamination was removed by DNAse I digestion before RT-PCR. CDNA was generated from 5 μg of total RNA using a random hexamers approach and RevertAid H Minus First Strand cDNA Synthesis kit (Life Technologies, Monza, Italy).
Gene expressions levels of sFlt-1, TNF-α, and IL-6 were determined by real-time PCR using specific TaqMan primers and probes (Life Technologies, Monza, Italy). MRNA levels were normalized using endogenous 18 s as internal reference (Life Technologies, Monza, Italy). Relative expression and fold change were calculated according to Livak and Schmittgen [64 (link)].
Gene expressions levels of sFlt-1, TNF-α, and IL-6 were determined by real-time PCR using specific TaqMan primers and probes (Life Technologies, Monza, Italy). MRNA levels were normalized using endogenous 18 s as internal reference (Life Technologies, Monza, Italy). Relative expression and fold change were calculated according to Livak and Schmittgen [64 (link)].
5
Quantitative Analysis of Placental Gene Expression
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Total RNA was extracted from frozen placental biopsies and explants using TRI reagent (Sigma-Aldrich, Milano, Italy) according to manufacturer instructions and next treated with DNAse I to remove genomic DNA contamination. Three µg of total RNA were reverse transcribed using a random hexamers approach (Fermentas Europe, St. Leon-Rot., Germany). Gene expression levels of HMGB1, IL-6 and TNFα were quantified by Real-time PCR using specific TaqMan primers and probes following manufacturer’s protocol (Life Technologies). For the relative quantitation, PCR signals were compared among groups after normalization using ribosomal 18S RNA expression as internal reference (Life Technologies). 18S mRNA expression was not affected by LMWH treatment. Relative expression and fold change were calculated according to Livak and Schmittgen [69 (link)].
6
Placental Gene Expression Analysis
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Total RNA was isolated from frozen placental biopsies using TRI® reagent (Sigma-Aldrich, Milan, Italy) according to the manufacturer’s instructions and then treated with DNase I to remove genomic DNA contamination. Three micrograms of total RNA was reverse-transcribed using a random-hexamer approach (Fermentas Europe, St. Leon-Rot, Germany) and a RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Cat. No k1632, Leon-Rot, Germany). qRT-PCR reactions were run on a StepOne™ real-time PCR system instrument (Applied Biosystems, Waltham, MA, USA). Gene expression levels of hypoxia-inducible factors 1 α (HIF-1α), CAT, and SOD1 were determined by real-time PCR using specific TaqMan primers and probes following the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA, Cat. No 4331182). TaqMan primers and probes for ribosomal 18S, HIF-1α, CAT, and SOD1 were purchased from Applied Biosystems as TaqMan gene expression assays. For relative quantification, PCR signals were compared between the groups after normalization using ribosomal 18S RNA expression as an internal reference (Life Technologies, Carlsbad, CA, USA, Cat. No 4333760F). Relative expression and fold change were calculated according to Livak and Schmittgen [34 (link)].
7
Gene Expression Analysis of Aorta Tissue
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The aorta branches were excised, cleaned, and homogenized with TRIzol reagent (Invitrogen) for RNA extraction using the Qiagen RNeasy kit. The cDNA was synthesized using BIO-RAD iScript reverse transcription supermix according to the manufacturer’s instructions. Quantitative real-time PCR was performed on BIO-RAD CFX96 Real-Time System thermocycler using specific TaqMan primers and probes (Life Technologies). Gapdh was used as the housekeeping gene to normalize the expression of the target genes, and the WT HFC-treated mice Ct was used in the delta delta Ct calculations for the determination of the fold gene expression. The primers (Assay ID) were as follows: Gapdh, Mm99999915_g1; IL-1β, Mm00434228_m1; Tnf-α, Mm00443258_m1; CCL2, Mm00441242_m1; CXCL1, Mm04207460_m1; CXLC2, Mm00436450_m1; IL-17A, Mm00439618_m1 (Thermo Fisher Scientific, Waltham, MA, USA).
8
Placental Gene Expression Quantification
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In parallel, total RNA was isolated from frozen placental biopsies using TRI reagent (Sigma-Aldrich, Milano, Italy) according to manufacturer instructions and next treated with DNAse I to remove genomic DNA contamination. 3 µg of total RNA were reverse transcribed using a random hexamers approach (Fermentas Europe, St. Leon-Rot., Germany). Gene expression levels of PlGF and sFlt1 were quantified by Real-time PCR using specific TaqMan primers and probes following manufacturer’s protocol (Life Technologies). TaqMan primers and probes for ribosomal 18S and PlGF were purchased from Applied Biosystems as TaqMan Gene Expression Assays. sFlt-1 primers and probe were designed as previously described by Nevo et al.68 (link) and purchased from Applied Biosystems as Custom Gene Expression Assays. For the relative quantitation, PCR signals were compared among groups after normalization using ribosomal 18S RNA expression as internal reference (Life Technologies) whose expression remains stable across patients. Relative expression and fold change were calculated according to Livak and Schmittgen69 (link).
9
Relative mRNA Quantification via qPCR
10
Evaluating Gene Expression in LNCaP Cells
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