Gene expression studies were performed as previously detailed (30 (link)). Total RNA was extracted using the PureLink™ RNA Mini kit (Life technologies, Carlsbad, CA, USA) according to manufacturer recommendations and quantified by absorbance at A260/A280 nm (ratio >1.95) in a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). After it was reverse-transcribed with SuperScript VILO Master Mix (Life technologies), the levels of the genes of interest were normalized for the housekeeping gene beta actin and quantified by the 2−ΔΔCt method in a VIIA7 instrument (Life technologies). The primers/ probe for the analyzed genes were purchased from Taqman® Assay on-demand library (Life technologies).
Viia7 instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The ViiA7 instrument is a real-time PCR system designed for quantitative gene expression analysis, genotyping, and copy number variation detection. The instrument utilizes a 96-well block format and offers multi-color detection capabilities.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (23 mentions)
Trizol reagent, by Thermo Fisher Scientific (15 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (7 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Viia7, by Thermo Fisher Scientific (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (5 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (5 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Taqman genotyping master mix, by Thermo Fisher Scientific (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Perfecta fastmix 2, by Quantabio (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
129 protocols using viia7 instrument
1
Gene Expression Analysis Using PureLink RNA Kit
2
RNA Extraction and Quantitative RT-PCR
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As previously detailed [13 (link),14 (link),16 (link),17 (link),18 (link)], total RNA was extracted using the PureLinkTM RNA Mini kit (Life technologies, Carlsbad, CA, USA) and quantified by absorbance at A260/A280 nm (ratio >1.95) in a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA). For quantitative RT-PCR experiments, 1.5 μg total RNA was reverse-transcribed with SuperScript VILO Master Mix (Life technologies). Messenger RNA levels of genes of interest were amplified, quantified, and normalized for the reference gene beta actin in a VIIA7 instrument (Life technologies).
3
Real-Time PCR Array for Gene Expression
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RealTimePCR array reactions were performed as previously described [41 (link)–43 (link)]. The extraction of total RNA from the remaining alveolus was performed with the RNeasyFFPE kit (Qiagen Inc, Valencia, CA) according to the manufacturers’ instructions. The integrity of the RNA samples was verified by analyzing 1 mg of total RNA in a 2100Bioanalyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturers’ instructions, and the complementary DNA was synthesized using 3 μg of RNA through a reverse transcription reaction (Superscript III, Invitrogen Corporation, Carlsbad, CA, USA). RealTimePCR array was performed in a Viia7 instrument (LifeTechnologies, Carlsbad, CA) using a custom panel containing targets "Wound Healing" (PAMM-121), "Inflammatory cytokines and receptors"(PAMM-011) and "Osteogenesis" (PAMM-026) (SABiosciences, Frederick, MD) for gene expression profiling. RealTimePCR array data was analyzed by the RT2 profiler PCR Array Data Analysis online software (SABiosciences, Frederick, MD) for normalizing the initial geometric mean of three constitutive genes (GAPDH, ACTB, Hprt1) and subsequently normalized by the control group, and expressed as fold change relative to the control group; as previously described [44 (link), 45 (link)].
4
Quantitative Real-Time PCR Protocol
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Real-time quantitative PCR was performed for each technical triplicate using the Power SYBR Green Master Mix (Life Technologies) with a VIIA7 instrument (Life Technologies). The program used consisted of the denaturation of the samples at 95 °C for 2 min, followed by 40 cycles of amplification (30 s of denaturation at 95 °C, annealing at the primer-specific temperature (57 °C–63 °C) for 30 s, and extension at 72 °C for 30 s). The primers were purchased from MWG, and the specific sequences, as well as the amplicon sizes, are provided in the Supplementary Material (Table S2 ). For the analysis of the qRT-PCR data, the housekeeping gene encoding ribosomal protein L37A was used to normalize the values of the tested genes. The expression levels were calculated using the ΔΔCT method and are shown as the mean value with the standard error of mean. The procedures used for RNA isolation and cDNA synthesis are described in the Supplementary Methods .
5
Quantification of mRNA Expression via RT-PCR
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mRNA expression was quantitated using RT-PCR as previously described [5 (link),46 (link),47 (link),48 (link)]. Briefly, TRIzol (Life Technologies) extracted total RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Total RNA (250 ng) was subjected to cDNA synthesis using the High Efficiency cDNA Synthesis Kit (Life Technologies). The levels of various mRNA transcripts were determined using Fast SYBR Green (Life Technologies) and gene-specific forward and reverse primer sets (Table 1 ) on a ViiA7 instrument (Life Technologies). 18s RNA expression was used to normalize mRNA expression using the ΔΔCT comparative method.
6
Developmental Gene Expression Profiling
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Total RNA from developmental stages between 16 and 32 cells, up to 120 hour post‐fertilization (hpf) was extracted using the RNeasy Mini kit according to the manufacturer's instructions (QIAGEN) using at least 50 embryos at each stage. The heart, liver and brain were dissected from five adult fish, flash‐frozen on dry ice and stored at −80°C until the RNA was extracted. Eyes were dissected (N = 40 eyes total) at 48 hpf stage and flash frozen on dry ice.
The PrimeScript RT reagent kit (Takara) was used to transcribe the RNA into the cDNA following the manufacturer's protocol. The presence of kiaa0319 transcripts was verified by electrophoresis following PCR amplification. Gene expression was assessed by quantitative PCR (qPCR) conducted with the Luna Universal RT‐qPCR Kit (NEB) and using a Viia7 instrument (Life Technologies, Paisley, UK). For protocol details seeSupplementary materials . Primer sequences and accession numbers are shown in Table S1. Differences in gene expression were evaluated with the Wilcoxon‐Mann–Whitney test implemented in R (RStudioTeam, 2015 ).
The PrimeScript RT reagent kit (Takara) was used to transcribe the RNA into the cDNA following the manufacturer's protocol. The presence of kiaa0319 transcripts was verified by electrophoresis following PCR amplification. Gene expression was assessed by quantitative PCR (qPCR) conducted with the Luna Universal RT‐qPCR Kit (NEB) and using a Viia7 instrument (Life Technologies, Paisley, UK). For protocol details see
7
CDKN2A Promoter Methylation Analysis
8
Validating Microarray Findings by qRT-PCR
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We validated microarray results for 14 out of 23 genes significantly changed in SRvsSS comparison by qRT-PCR as previously described15 . Briefly, we reversed-transcribed individual total RNA from 6 rats per group into cDNA using Advantage RT-for-PCR kit (Clontech, Mountain View, CA, USA). We generated gene-specific PCR primers using LightCycler probe design software v. 2.0 (Roche Biosystems, Indianapolis, IN, USA) and purchased primers from the Synthesis and Sequencing Facility of Johns Hopkins University (Baltimore, MD, USA). We used High Capacity cDNA Reverse Transcription Kit (Invitrogen, Waltham, MA, USA) with a ViiA 7 instrument (Life Technologies, Waltham, MA, USA) to perform PCR. We normalized the relative amounts of mRNA in each sample to means of clathrin, ornithine decarboxylase antienzyme 1, and tubulin.
9
qPCR Analysis of CYP3A4 and miRNA Expression
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Total RNA (500 ng) was reverse transcribed to cDNA using PrimeScript RT Reagent kits (Takara Bio, Dalian, China). The relative expression of CYP3A4 mRNA was measured by qPCR using the appropriate primers (forward, 5′-CCAAGCTATGCTCTTCACCG-3′; reverse, 5′-TCAGGCTCCACTTACGGTGC-3′) and the FastStart Universal SYBR Green Master (Roche, Germany) according to the manufacturer's protocol. GAPDH mRNA was quantified as the endogenous control using the appropriate primers (forward, 5′-ATCACCATCTTCCAGGAGCGA-3′; reverse, 5′-GCTTCACCACCTTCTTGATGT-3′). For both sets of qPCR primers, the forward primer and the reverse primer were designed to target different exons to avoid the possibility of amplifying genomic DNA. The specificity of the qPCR primers was verified by the presence of a single peak in the melting curves. For miRNA quantification, reverse transcription and qPCR were performed using the miRCURY LNA™ Universal RT microRNA PCR system (Exiqon, Denmark) following the manufacturer's protocol. The U6 snRNA was measured as the endogenous control. All qPCRs were run on a ViiA 7 instrument (Life Technologies) with three replicates. The Cq values were determined automatically by the ViiA 7 RUO Software v1.1 (Life Technologies). All NTCs' Cq values were higher than 40 and were labeled as “undetermined” by the ViiA 7 RUO Software v1.1.
10
Fibroblast Gene Expression Analysis
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Fibroblasts were plated on 50 µg/mL of BME and treated with 10 µM of i-bodies or 12 µM of AMD3100. After 48 h, RNA was extracted using Trizol reagent and reverse transcribed into cDNA using superscript II reverse transcriptase (Life Technologies) as previously described29 (link). Complementary DNA (cDNA) was subsequently loaded into a Taqman plate and gene expression analysis were performed using predesigned primers for ACTA2, COL1A1, COL3A1 and FN1. All Taqman analysis was performed using Applied Bio system’s Viia 7 instrument (Life Technologies). The results were then exported, normalized to 18S RNA expression and fold change analyses were calculated using Data Assist software (Life Technologies).
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