For quantitative real-time PCR, RNAs were reverse-transcripted to cDNA using the Invitrogen SuperScript VILO™ Master Mix (No. 11755, Life Technologies). The expression of genes coding for known pro- and anti-angiogenic factors [25] (link), inflammatory factors, the glial cell marker and endogenous control genes (Table 1
Taqman array 96 well fast plate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan array 96-well FAST plate is a preformatted microfluidic plate designed for real-time PCR analysis. It allows for the simultaneous detection and quantification of multiple gene targets in a single sample across 96 wells.
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Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Stepone plus real time polymerase chain reaction system, by Thermo Fisher Scientific (2 mentions)
Expressionsuite software ver 1.3, by Thermo Fisher Scientific (2 mentions)
Dataassist software v3, by Thermo Fisher Scientific (1 mentions)
Stepone software v2, by Thermo Fisher Scientific (1 mentions)
1xtaqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex instrument, by Thermo Fisher Scientific (1 mentions)
Nanovue plus spectrophotometer, by GE Healthcare (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent solution, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
15 protocols using taqman array 96 well fast plate
1
Quantitative Real-Time PCR for Angiogenic Factors
2
Evaluating Gene Expression Profiles of MSCs
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The effects of γ-irradiation and starvation culture on gene expression of MSCs (three different samples) and two control cell lines (A549 and HCC1937) were assessed using custom Taqman® array 96-well Fast plates (Applied Biosystems, Life Technologies), previously designed to measure the expression level of genes involved in DDR pathway and cell cycle (47 target genes plus 1 endogenous control, in duplicate). Briefly, after irradiation and starvation culture, RNA was isolated with TRIzol (Life Technologies) followed by phenol-chloroform extraction and reverse transcription. Then, 25 ng of cDNA per well of each sample were plated in custom array plates (each well corresponding to one gene assay) and quantitative real-time PCR was performed by means of Quantstudio 12K Flex instrument (Life Technologies). Data were analyzed with Quantstudio Analysis software (Applied Biosystem). Statistical analysis was performed using paired Student's t-test.
3
Quantitative Gene Expression Analysis of Intervertebral Disc Degeneration
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Total RNA, from NP and iAF cells from non-degenerating and degenerating IVDs of five additional individuals was isolated, and reverse transcribed as previously described [50 (link),141 (link)]. Target genes were selected and analyzed (Tables S24 and S25 ). Custom TaqMan Array 96-Well Fast Plates (Applied Biosystems, Foster City, CA, USA) were designed to analyze the selected genes using the primers described in Table S23 . The expression of mRNAs was determined using TaqMan Master Mix (2×), and qPCR data analysed from cDNA arrays by the 2−DDCT method [146 (link)] was used to validate candidate genes identified through scRNA-Seq and the validation by protein analysis.
4
Gene Expression Analysis in Cancer Cell Lines
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SCC-9 and FaDu cells were cultured on 10cm 2 dishes and incubated overnight. After treatment, cells were washed, and RNA was extracted and purified with TRIzol (Invitrogen) + chloroform (0.2 ml/1 ml TRIzol) and the PureLink RNA Mini Kit (Life Technologies), following the manufacturer's instructions. Samples were treated with the Turbo DNA-free kit (Life Technologies) and stored at -20 C.
RNA was quantified and had its quality assessed on a NanoVue Plus spectrophotometer (GE Health Care, Little Chalfont, UK). A total of 2 μg RNA was diluted to the concentration of 0.1 μg/μl, submitted to the reverse transcription reaction using the SuperScript VILO MasterMix (Invitrogen), and stored at -20 C. cDNA samples were diluted to the concentration of 12.5 ng/μl, and 5 μl (12.5 ng cDNA) was mixed to 5 μl TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, California) per reaction. This PCR reaction mix was added to each well of custom TaqMan Array 96-well FAST plates (Applied Biosystems) that were designed for this experiment (Table S1). The plates were submitted to the PCR on a StepOnePlus Real-Time PCR System (Applied Biosystems), according to the thermal cycle conditions suggested by the TaqMan MasterMix protocol.
RNA was quantified and had its quality assessed on a NanoVue Plus spectrophotometer (GE Health Care, Little Chalfont, UK). A total of 2 μg RNA was diluted to the concentration of 0.1 μg/μl, submitted to the reverse transcription reaction using the SuperScript VILO MasterMix (Invitrogen), and stored at -20 C. cDNA samples were diluted to the concentration of 12.5 ng/μl, and 5 μl (12.5 ng cDNA) was mixed to 5 μl TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, California) per reaction. This PCR reaction mix was added to each well of custom TaqMan Array 96-well FAST plates (Applied Biosystems) that were designed for this experiment (Table S1). The plates were submitted to the PCR on a StepOnePlus Real-Time PCR System (Applied Biosystems), according to the thermal cycle conditions suggested by the TaqMan MasterMix protocol.
5
Transcriptomic Analysis of Lipid Metabolism and ABC Transporters
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Total RNA from B16-F10 and NIH/3T3 cells was isolated using TRI Reagent Solution™ (Invitrogen™). Reverse transcription was performed using the High Capacity RNA-to-cDNA kit (Applied Biosystems™) according to the manufacturer's instructions. Gene expression was investigated with TaqMan ® Array 96-WELL FAST Plates (Applied Biosystems™): mouse lipid regulated genes (cat no: 4415461) and mouse ABC transporters (cat no: 4418752). All samples were analysed in duplicate using 100 ng total RNA/sample. The relative gene expression was calculated using the ∆∆Cq method. The results were analyzed using the ExpressionSuite Software v 1.1.
6
Quantitative Gene Expression Analysis
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Total RNAs were extracted using the NucleoSpin RNA II kit (Macherey-Nagel) and cDNA synthesized using the QuantiTect reverse transcription kit (Qiagen) following the manufacturer's recommendations. qPCR analysis was performed with custom TaqMan® Array 96-Well Fast plates (Thermo Fisher Scientific) according to the manufacturer's protocol. All primers and MGB probes labeled with FAM for amplification were purchased from Thermo Fisher Scientific (Table S2 ). Results were normalized against 18S, and quantification of gene expression was based on the deltaCt method in minimum three independent biological experiments.
7
qPCR Gene Expression Analysis
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Total RNAs were extracted using RNeasy PlusMini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. RNA yields and quality were checked with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 2 μg of mRNA using SuperScript™ VILO™ MasterMix (Thermo Fisher Scientific) following the manufacturer’s recommendations. Synthesized cDNA was diluted at 1/9 in DNase-free water before performing quantitative PCR. qPCR analysis was performed on QuantStudioTM 12K Flex Real-Time PCR System (Thermo Fisher Scientific) with custom TaqMan® Array 96-Well Fast plates and TaqMan® Gene expression Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. All primers and Minor Groove Binder (MGB) probes labeled with fluorescein (FAM) for amplification were purchased from Thermo Fischer Scientific (Table S2 ). Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and quantification of gene expression was based on the Delta Delta Ct Method in three independent biological experiments.
8
Quantification of Immune Genes in Kidney
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Total RNA was extracted from kidney tissues using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions and quantitated using a NanoDrop 2000c spectrophotometer (Thermo Scientific). Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) with TaqMan Array 96-Well Fast Plates containing specific primers and Universal Master Mix II (Life Technologies) to evaluate the gene expression of IL-1β (Mm00434228_m1), IL-2 (Mm00434256_m1), IL-6 (Mm00446190_m1), IL-10 (Mm00439614_m1), IL-18 (Mm00434225_m1), TNF-α (Mm00443258_m1), IFNα (Mm04207507_gH), ICAM1 (Mm00516023_m1), C-X-C motif chemokine 5 (Mm00436451_g1), HMGB1 (Mm00849805_gH), RAGE (Mm01134790_g1), TLR4 (Mm00445273_m1), TLR7 (Mm00446590_m1), TLR9 (Mm00446193_m1), and NACHT, LRR, and PYD domains containing protein 3 (Mm00840904_m1). The relative mRNA abundance was standardized using GAPDH and 18S mRNA as the invariant control.
9
Quantitative RNA Expression Analysis by RT-qPCR
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Total RNA was extracted using a Nucleospin RNA II kit (Macherey-Nagel) according to the manufacturer’s protocol and RNA yields and quality assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized from 100 ng total RNA using the QuantiTect reverse transcription kit (Qiagen) following the manufacturer’s recommendations. Synthesized cDNA was then diluted 1/20 in DNase-free water before performing real-time quantitative PCR (RT-qPCR). RT-qPCR analysis was performed using an Applied Biosystems real-time PCR device (7500 Fast System) with custom TaqMan® Array 96-Well Fast plates and TaqMan® Gene expression Master Mix (Life Technologies) following the manufacturer’s instructions. All primers and MGB probes labeled with FAM for amplification were purchased from Life Technologies (Supplementary Data 1 ). Results were normalized against those obtained with 18 S rRNA and the quantification of gene expression was based on the delta-deltaCt method (Eq. 1 ) in three minimum independent biological experiments. where Ct = cycle threshold; t = time; n = independent biological experiments.
10
RNA Expression Analysis of Fibrous Capsules
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RNA was extracted from the recipient subcutaneous fibrous capsules surrounding the silicone spacer at 2, 4, 6 and 8 weeks after pretreatment29
(2W, n = 5; 4W, n = 5; 6W, n = 5; 8W, n = 5). The relative gene expression was determined using a TaqMan array 96-well FAST plate (4413257; Applied Biosystems, Bedford, MA, USA). A TaqMan array plate contains 46 target genes and two assays for candidate endogenous control genes (Table 1 ). The samples were analyzed using a StepOnePlus Real-Time polymerase chain reaction (PCR) System (Applied Biosystems) under the following amplification conditions: 50°C for 2 min and 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The results were analyzed using ExpressionSuite Software (ver. 1.3; Applied Biosystems). Relative quantification (RQ) was calculated using the comparative CT method. To determine the relative gene expression in the 6W group, the samples in the 2W group were designated as a calibrator. 18S was used as a housekeeping gene.
(2W, n = 5; 4W, n = 5; 6W, n = 5; 8W, n = 5). The relative gene expression was determined using a TaqMan array 96-well FAST plate (4413257; Applied Biosystems, Bedford, MA, USA). A TaqMan array plate contains 46 target genes and two assays for candidate endogenous control genes (
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