One cubic millimetre punches of striatal tissue at the level of the injection were removed for RNA extraction. RT-PCR assays were performed as previously described [30 (link)]. Samples were run against absolute copy number standard curves generated from serially diluted cDNA from LPS-challenged rat liver. Primer and probe sets for rat IL-1β, CXCL-1, CCL-2, CXCL-10, CCL-3 (MIP-1α), and CCL-4 (MIP-1β) were designed using the Roche universal probe library assay design centre (Roche.com). Samples were analysed using a Roche Light Cycler 480® (Roche Diagnostics, Welwyn Garden City, UK) and all reagents were used according to manufacturer's instructions. Briefly, gene-specific primers were combined with a FAM/TAMRA labelled hybridisation probe (for sequences see Additional file 1 : Table S1). PCR was run according to standard conditions [31 (link)]. Analysis was performed using the Pfaffle method, taking the standard curve to determine reaction efficiency followed by a comparative-cycle-threshold method. Results are expressed as absolute copy number.
Universal probe library assay design centre
Manufactured by Roche
Sourced in Germany
The Universal Probe Library Assay Design Centre is a comprehensive online platform that provides tools and resources for the design of real-time PCR assays. It offers access to a collection of pre-designed, validated probes that can be used in various gene expression analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (2 mentions)
7900 sequence detector, by Thermo Fisher Scientific (2 mentions)
Faststart universal probe master mix, by Roche (2 mentions)
S lycopersicum oligonucleotide primers, by Merck Group (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Supermix with premixed rox dye, by Thermo Fisher Scientific (1 mentions)
Universal probe library set mouse, by Roche (1 mentions)
Prism 7, by GraphPad (1 mentions)
Facsaria iiitm, by BD (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Purelink rna micro scale kit, by Thermo Fisher Scientific (1 mentions)
Agilent rna 6000 pico assay, by Agilent Technologies (1 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
15 protocols using universal probe library assay design centre
1
Quantitative RT-PCR Analysis of Inflammatory Markers
2
Quantitative PCR for CUL4A Expression
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Total RNA was extracted from three independent plates for each cell line (RNAeasy Kit, Quiagen, Valencia, CA) and converted to cDNA by using 500ng as template (High Capacity cDNA RT kit, Applied Biosystems, Foster City, CA). qPCR assays were designed for target gene (CUL4A) and endogenous control (β-ACTIN ) using the Roche Universal Probe Library Assay Design Centre web site (Roche Applied Science, Indianapolis, IN) (Supplementary Table S2 ). Reactions were performed in triplicate using the ABI Prism 7900HT Sequence Detection System according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). Relative expression was determined using the qBase software that allows for PCR efficiency correction and implements normalization by endogenous genes [46 (link)].
3
Quantitative RT-PCR Protocol for Gene Expression
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Quantitative real-time PCR (TaqMan method) was performed using SuperMix with Premixed ROX dye (Invitrogen), primer sets were designed using the Roche Universal Probe Library Assay Design Centre and purchased from Eurofins MWG Operon (Ebersberg, Germany) and probes from the Roche Universal Probe Library Mouse Set (Roche Applied Science). Samples were assayed in duplicate and run on an ABI 7900HT Fast Real-Time PCR machine using the following conditions: 95°C for 10 minutes then 40 cycles of 95°C for 15 s and 60°C for 1 min. Primer amplification efficiency was validated, and analysis was performed using the relative standard curve method. Data were normalised to Actb and fold-change is expressed as the ratio of expression of each gene of interest in the treated groups against the average of the VC groups. Statistical analysis was performed using GraphPad Prism 7.0. Data are presented as mean ± s.e.m. and statistical comparisons are described in figure legends. Criterion for significance was P < 0.05. Primer pair and probe information is provided in Supplementary Table 1 (see section on supplementary data given at the end of this article).
4
Profiling Cell-Specific Gene Expression
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CD19+ B cells, CD3+ T cells, CD14+ monocytes, and CD56−HLA‐DR+ dendritic cells were sorted using a high‐speed cell sorter (FACSAria IIITM; BD Biosciences), resulting in a purity of more than 95%. Subsequently, mRNA was isolated using GenEluteTM Mammalian RNA Kit (Sigma‐Aldrich, St. Louis, MO) and reversely transcribed into cDNA following a standard laboratory protocol with the use of SuperScript II® Reverse Transcriptase (Invitrogen, Paisley, UK). Primers and probes were selected by using the Universal Probe Library Assay Design Centre (Roche Applied Science, Penzberg, Germany). To determine target gene mRNA expression levels, qPCR was performed using an Applied Biosystems 7900 Sequence Detector, which was programmed for the initial step of 2 min at 50°C and 10 min 95°C, followed by 40 thermal cycles of 15s at 95°C and 1 min at 60°C. For the calculation of relative mRNA levels, CT values per gene were related to standard curves, which were generated for each gene of interest. The 18S levels were measured as a control to normalize for RNA input. Primer sequences are listed in Supporting Information Table 3 .
5
Transcriptome Analysis of Enriched Cell Populations
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Total RNA was isolated from pellets of enriched cell populations using the PureLink RNA Micro Scale Kit (Thermo Fisher Scientific, Schwerte, Germany). An on‐column DNase digestion step (PureLink DNase mixture, Thermo Fisher Scientific, for 20 min at RT) was included to remove genomic DNA (Roche Molecular Systems, Mannheim, Germany). RNA integrity was validated using the Agilent RNA 6000 Pico Assay according to the manufacturer's instructions (Agilent Technologies, Waldbronn, Germany). First‐strand cDNAs from the total RNA purified from each cell population were synthesized using the RevertAid H Minus First‐Strand cDNA Synthesis Kit (Fermentas by Thermo Fisher Scientific, Schwerte, Germany). Primers were designed using the Universal ProbeLibrary Assay Design Centre (Roche, Table S2 ) and transcript levels of candidate genes were measured by qRT‐PCR using the TaqMan hPSC Scorecard Panel (384 well, ViiA7, Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions.
6
Quantitative RNA Expression Analysis
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For isolation of total RNA 5-7 cryo-sections of 50 μm were used. RNA isolation was performed by using the GenElute Mammalian Total RNA Miniprep Kit (Sigma). RNA samples were treated with DNAse I to remove contaminating DNA (Invitrogen). Using 1.0 μg RNA as a template, copy DNA (cDNA) was reverse transcribed by using Superscript II (Invitrogen). Primers and probes were selected by using the Universal ProbeLibrary Assay Design Centre (Roche). To determine target gene mRNA expression, real-time quantitative reverse transcription PCR was performed using TaqMan technology. GAPDH mRNA and GUSB (for brain tissue) or 18S (for astrocyte cultures) RNA levels were measured as a control to normalize for RNA input. Both GUSB and 18S validated the GAPDH results. Reference gene primers and probes were obtained from Applied Biosystems. An Applied Biosystems 7900 Sequence Detector was programmed for the initial step of 2 min at 50°C and 10 min at 95°C, followed by 40 thermal cycles of 15 s at 95°C and 1 min at 60°C. For calculation of mRNA expression levels, Ct values per gene were applied to standard curves, generated for each gene of interest. Sequences of the primers are listed in Additional file 1 : Table S1a.
7
Quantitative RT-PCR Analysis of Gene Expression
8
Quantitative PCR analysis of C. elegans outrons
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Total RNA was prepared from C. elegans strains grown on E. coli using the PureLink RNA Mini kit (Life Technologies) with modifications for TRIzol treated samples and DNAse treatment as described by the manufacturer. For cDNA synthesis, total RNA was reverse transcribed using oligo(dT) Primers and M-MLV Reverse Transcriptase, according to the manufacturer's instructions. qPCR assays were designed using the Universal Probe Library Assay Design Centre (Roche) and tested for efficiency (Supplementary Table S2 ). qPCR was done in three technical replicates on a Roche Lightcycler 480 using standard settings. The Minimum Information for Quantitative Real-Time PCR Experiments (MIQE) is included as Supplementary Data . Data was analyzed using the comparative CT method, assuming a primer efficiency of 2 (27 (link)). Outron levels were standardized with respect to internal RNA. Outron levels detected in unc-22(RNAi) treated animals were defined as 1.
9
Quantitative PCR Gene Expression Analysis
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The Applied Biosystems TaqMan method (Thermo Fisher Scientific) was used to detect specific PCR products. Primers for genes of interest were designed by the Universal Probe Library Assay Design Centre (Roche Applied Science, Penzberg, Germany) and purchased from Eurofins MWG Operon (Ebersberg, Germany) (sequences in Table S3 ). Reactions were prepared in duplicate and amplification performed at 95°C for 5 minutes and then, 30 cycles of 95°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute on a real time PCR system (Quantstudio5). Relative expression for each gene was calculated using the standard curve method in which the amount of target genes was normalized to beta‐actin (Actb) and relative expression between samples was calculated. Normalized expression values are displayed as a fold change in expression. Statistical analyses were performed using GraphPad Prism software. When data were normally distributed a student's t test was performed to determine the significance of a difference between two groups. When comparing the means of more than two groups a one‐way ANOVA was used followed by a multiple comparisons test such as Sidak's or Tukey's. All data are presented as mean ± SEM and criteria for significance is P < .05.
10
RNA Isolation, cDNA Synthesis, and qPCR Analysis
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RNA was isolated from BMDMs and BMDCs using RNeasy Micro Kit (Qiagen cat. 74004) which included DNase treatment. Total RNA was quantified using Qubit RNA high sensitivity kit (Biosciences cat. Q32852). Complementary DNA (cDNA) was synthesized from 100 ng of total RNA using Transcriptor Reverse Transcriptase (Roche) and random hexamer primers (Roche) as per manufacturer’s instructions. qPCR assays were designed using the Roche Universal Probe Library Assay Design Centre (Table 2 ). qPCR assays were carried out by using SensiFAST Probe No-ROX Kit (Bioline), 500 nM primers, 250 nM corresponding ProbeLibrary probe from Universal ProbeLibrary (Roche) and 2 ng of cDNA. PCR reactions were run on a LightCycler 480 instrument (Roche) and the 2–ΔΔCT method (Mar et al., 2009 (link); McCall et al., 2014 (link)) was used to calculate relative gene changes in gene expression compared to β-actin which served as a housekeeping gene (Livak and Schmittgen, 2001 (link)).
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