RNA was extracted from lung tissue using Trizol (Invitrogen) and the RNeasy kit (Qiagen). Quantitative RT-PCR was performed using the Omniscript reverse transcription kit, and amplification was performed using the Veriquest PCR master mix and specific Taqman probes from Applied Biosystems. Expression relative to the human RPL7 or mouse Rpl7 ‘housekeeping’ gene was calculated using the Delta Delta Ct method. RNA sequencing was performed using the KAPA Stranded mRNA-Seq Library Preparation kit (Kapa Biosystems) following the manufacturer’s instructions using an Illumina HiSeq 2000 or HiSeq 2500 platform (Illumina, San Diego, CA). Sequenced reads (50 bp, single end) were mapped to the mouse genome (NCBI37/mm9, July 2007) using Bowtie 0.12.9, and only the reads that mapped onto exons of each RefSeq gene were measured and normalized using reads per kilobase per million mapped reads. Analyses of differential gene expression were performed using R package edgeR.
Specific taqman probes
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan probes are targeted DNA sequences that are used in real-time PCR (polymerase chain reaction) assays. They are designed to detect and quantify specific nucleic acid targets. TaqMan probes contain a fluorescent reporter dye at one end and a quencher dye at the other end. When the probe is intact, the quencher dye suppresses the fluorescence of the reporter dye. During the PCR process, the probe binds to its complementary target sequence, and the fluorescent reporter dye is cleaved, emitting a fluorescent signal that can be detected and measured.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (9 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (4 mentions)
High capacity cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Specific taqman primers, by Thermo Fisher Scientific (4 mentions)
Mirneasy kit, by Qiagen (4 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Quantitect reverse transcription kit, by Qiagen (3 mentions)
Rneasy kit, by Qiagen (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Dynamo probe qpcr kit, by Thermo Fisher Scientific (2 mentions)
Cd90.1 microbeads, by Miltenyi Biotec (2 mentions)
Qiashredder column, by Qiagen (2 mentions)
48 protocols using specific taqman probes
1
Transcriptome Analysis of Lung Tissue
2
Comprehensive RNA Analysis Pipeline
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Total RNA was isolated from eWAT and liver using the miRNeasy kit (Qiagen, Hilden, Germany) and assessed for quality (Agilent Bioanalyzer). Samples with RIN < 7 were excluded (n = 1 for WT-HFD eWAT and liver). For gene expression analysis, cDNA was synthesized from 200 ng of total RNA with the High-Capacity cDNA Synthesis kit (Applied Biosystems, Foster City, CA). qRT-PCR was performed using the 2x Fast SYBR Green Master Mix (Applied Biosystems; primer sequences in Supplementary Table 1 ). miRNAs were reverse-transcribed with specific TaqMan primers (Applied Biosystems), and subsequently measured by real-time PCR with the TaqMan universal PCR master mix and specific TaqMan probes (Applied Biosystems).
3
RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted from myometrial tissue using TRI-Reagent (Sigma Aldrich, Germany) following the standard protocol. The details of RNA extraction and preparation for further analyses were described previously [19 (link)]. RNA precipitates were used for analyses that were integral electrophoretically (separation in 1.5% agarose gel) and possessed an optical density ration A260/A280 in the range of 1.8–2.0 (spectrophotometer Tecan, Männedorf, Switzerland). The Real-Time PCR was performed according to the guidelines provided by Bustin et al. (2009) [58 (link)]. The amplification was conducted using 4 pg/μL aliquots of extracted RNA, TaqMan®RNA-to- 1-Step Kit (Applied Biosystems, Foster City, CA, USA) and specific TaqMan probes provided by Applied Biosystems (Foster City, CA, USA) listed in Table 4 . Amplification was conducted in an AriaMX apparatus (Agilent Technologies, Santa Clara, CA, USA), with a standard thermal profile suggested by the producer. The Ct values were obtained with Aria 1.6 software (Agilent Technologies, Santa Clara, CA, USA) and used for cycle threshold (Ct) calculation. The Ct values of the tested genes were normalized with the geometrical mean of the reference genes and used for 2−∆∆Ct calculation [59 (link)].
4
Quantifying RNA Expression via qRT-PCR
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Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. PrimeScript RT Reagent kit (Takara) and SYBR Premix Ex Taq RT-PCR kit (Takara) were used to detect the quantities of mRNA expression. The fold-change of each gene was normalized to GAPDH as the internal control using 2–ΔΔCt method. TaqMan MicroRNA Assay kit (Applied Biosystems) and specific TaqMan probes (Applied Biosystems) were used to detect the expression of miRNAs. RNU6 was used as the internal control for humans, and sno202 was used as the internal control for mice.
5
Comprehensive RNA Analysis Pipeline
6
Quantification of miRNA Levels by RT-qPCR
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miRNAs levels were measured by RT-qPCR, 1.5 μL of isolated RNA was retrotranscribed with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). The RT reaction comprised a single cycle of 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C. Each miRNA was amplified from 2 μL of the RT (using specific primers) with the specific TaqMan probes (Applied Biosystems). Using the LightCycler TaqMan Master Kit reagent, PCR was carried out on a LightCycler 480 II thermocycler (Roche Applied Science, Basel, Switzerland). The PCR conditions included an enzyme activation step of 10 min at 95 °C, followed by 45 cycles of 95 °C for 15 s, at 60 °C for 60 s, and at 72 °C for 1 s. Relative miRNA levels was determined using the 2−Target miRNA Ct− cel-miR−39 Ct) equation.
7
Predicting miRNA Regulation of TGF-β2
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TargetScan 6.0 (http://www.targetscan.org/ ) was used to predict potential miRNAs binding to TGF-β2. The predicted miRNAs were tested using quantitative real-time (qRT)-PCR. For miRNA analysis, qRT-PCR was performed using TaqMan microRNA Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions with specific TaqMan probes (Applied Biosystems; Thermo Fisher Scientific, Inc.). For quantitation of mRNA, a Bio-Rad CFX96 Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) was employed according to the manufacturer's instructions. All mRNA and miRNA quantification data were normalized to GAPDH and U6, respectively. hTGF-β2 sense, 5′-TGGTGAAAGCAGAGTTCAGAG-3′ and antisense, 5′-CACAACTTTGCTGTCGATGTAG-3′; GAPDH sense, 5′-AGCCTCCCGCTTCGCTCTCT-3′ and antisense, 5′-GCGCCCAATACGACCAAATCCGT-3′; U6 sense, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and antisense, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; FOXD3 sense, 5′-GTCCGCTGGGAATAACTTTCCGTA-3′ and antisense, 5′-ATGTACAAAGAATGTCCCTCCCACCC-3′ were used
8
Quantifying Gene Expression in Adipose Tissue
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RNA from animal tissue samples (liver, mesenteric and epididymal fat) and 3T3-L1 adipocytes was extracted using Trizol (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following standard protocols. cDNA was obtained from 2 µg of RNA using M-MLV Reverse Transcription enzyme (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Gene expression was analysed using Taqman universal PCR Master Mix (Applied Biosystems, Cheshire, UK) and following a standard protocol: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of denaturing at 95 °C during 15 s plus an annealing/extension step at 60 °C for 1 min. Specific Taqman probes (Applied Biosystems, Cheshire, UK) were used for each gene: insulin receptor β (Ir-B, Mm_01211875_m1), caveolin 1 (Cav1, Mm_00483057_m1), Slc2a4 (Glut4, Mm_00436615_m1), and glyceraldehyde 3-phosphate dehydrogenase(Gapdh, Mm_05724508_g1). All reactions were performed in triplicate. Gapdh was used as invariant internal control for qPCR and subsequent normalization. Relative quantification followed 2−ΔΔCt method [54 (link)].
9
Transcriptomic analysis of mast cell activation
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BMBs were activated with either IgE/DNP or 10 ng/ml mIL-33 (R&D Systems) for 4 hours. mRNA was extracted by RNeasy kit (Qiagen) and cDNA was generated by qScript cDNA synthesis kit (Quanta BioSciences). Microarray assessment was performed by the Northwestern University Genomics Core using llumina MouseWG-6 Beadchips. Heatmaps were generated using GENE-E software (Broad Institute, http://broadinstitute.org/cancer/software/GENE-E ). Network diagrams were generated using the “FGNet” R package and the GeneTerm Linker algorithm (adjusted P value < 0.05; minimum support of 3). Visualization of these networks was performed with the “iGraph” R package and Cytoscape 3.2.1. Gene expression was also determined by real-time PCR using an ABI 7500 thermal cycler (Applied Biosystems) and specific TaqMan probes (Applied Biosystems) for each gene of interest. β-actin expression was used as an internal control, and changes in the threshold cycle values were determined.
10
Transcriptomic Response to Adrenergic Stimuli
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RNASeq experiments were done comparing untreated cells with a treatment with isoproterenol, adrenaline or noradrenaline for 4 h in THP1 cells.
qPCR was run in THP1 cells for 4 h incubation time with isoproterenol and formoterol at 1, 10 and 100 nM. Total RNAs were isolated with MagMAX™−96 Total RNA Isolation Kit (Ambion ref#AM1830), and cDNA was made using a cDNA Synthesis Kit (Applied Biosystems™ Ref#4368813) RT-PCRs were performed in 384-well plates on an AB7900HT cycler (Applied Biosystems) using specific TaqMan probes (Applied Biosystems). Housekeeper normalization was done relative to the one of the three genes GAPDH, PPIB or TBP, which had the most similar expression level to the gene of interest, according to our DMSO qPCR data. All measurements were done in quadruplicates.
qPCR was run in THP1 cells for 4 h incubation time with isoproterenol and formoterol at 1, 10 and 100 nM. Total RNAs were isolated with MagMAX™−96 Total RNA Isolation Kit (Ambion ref#AM1830), and cDNA was made using a cDNA Synthesis Kit (Applied Biosystems™ Ref#4368813) RT-PCRs were performed in 384-well plates on an AB7900HT cycler (Applied Biosystems) using specific TaqMan probes (Applied Biosystems). Housekeeper normalization was done relative to the one of the three genes GAPDH, PPIB or TBP, which had the most similar expression level to the gene of interest, according to our DMSO qPCR data. All measurements were done in quadruplicates.
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