Significantly altered genes from premanifest HD versus controls were selected from the SAM analysis to confirm affymetrix findings by RT-qPCR. Validations were initially carried out using RT-qPCR on samples from cohort 1; both the samples with RIN ≥ 5 used for above affymetrix and on all remaining samples: 5 premanifest (3 male, 2 female); 6 stage II/III HD patients (6 male, 0 female); and 7 control subjects (5 male, 2 female). Further validations were then performed on a separate cohort (cohort 2): 9 premanifest (5 male, 4 female); 9 stage II/III HD patients (5 male, 4 female); and 10 control subjects (5 male, 5 female) (See Tables 123). For RT-qPCR experiments, all samples were run in triplicate for each target gene and housekeeping gene, and relevant negative and positive controls were run on each plate. Melt curves were inspected for all assays, with the Tm checked to be within known specifications for each assay. Sample assay data points were included in data analysis only if detected with Ct < 37 and at least 3 Ct values lower than the corresponding negative control [16] . Any data that did not pass these criteria were omitted from all further analyses. Primers utilised for RT-qPCR validations (see Table 4) were designed using either QuantPrime [17] , Primer3 [18, 19] or PrimerQuest from Integrated DNA Technologies (http://eu.idtdna.com/PrimerQuest).
Primerquest
Manufactured by Integrated DNA Technologies
Sourced in United States
PrimerQuest is a web-based tool that assists users in designing high-quality PCR primers and probes for a variety of applications. The tool utilizes advanced algorithms to generate primers and probes that meet specific criteria, such as melting temperature, GC content, and specificity, to ensure optimal performance in PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Lightcycler 480 real time pcr system, by Roche (3 mentions)
Revertra ace qpcr rt kit, by Toyobo (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Lightcycler 480, by Roche (3 mentions)
Abi prism viia7, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Promega (3 mentions)
High capacity cdna reverse transcription kit, by Promega (3 mentions)
Quantprime, by Integrated DNA Technologies (2 mentions)
Cfx manager software program, by Bio-Rad (2 mentions)
Quantprime69, by Integrated DNA Technologies (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Masterpure dna purification kit for blood, by Illumina (2 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
87 protocols using primerquest
1
Validating Affymetrix Findings in Huntington's Disease
2
Validated RT-qPCR for Gene Expression
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SsoAdvanced Universal SYBR Green Supermix from Bio-Rad Laboratories was used for RT-qPCR and performed following manufacturer’s protocol. All RT-qPCR plates were run on a CFX96 touch real-time PCR detection system (Bio-Rad, CA, USA). Primers utilized for RT-qPCR validations (see Supplementary Table 1 ) were designed using either QuantPrime69 (link) or PrimerQuest from Integrated DNA Technologies (PrimerQuest">http://eu.idtdna.com/PrimerQuest ). The efficiency of each primer pair was tested before use by performing a standard curve, and the efficiency criteria for using a primer pair was 90% < E < 110%, with an R2 cut-off >0.990. Housekeeping genes, TATA-binding protein (Tbp) and 18S, were used for normalization of skeletal muscle tissue, and Glucuronidase beta, Gusb; Heat Shock Protein 90 kDa Alpha (Cytosolic), Class B Member 1, Hsp90ab1; and Tbp were used for normalization of adipose tissue RT-qPCR. Changes in gene expression were calculated using the CFX manager software program (Bio-Rad, CA, USA), using the ΔΔCt method with a fold change cut-off at ≥1.5 and p < 0.05 considered significant. All samples were run in triplicate and relevant positive and negative controls were run on each plate.
3
Quantifying Iron Regulon Gene Expression in Yeast
4
Sensitive Detection of Leptospira interrogans
5
Quantifying p300/CBP mRNA in Aphids
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Single aphids (n = 5) were collected 12 h post-injection (3000-ng/µL p300/CBP or GFP dsRNA) and RNA was extracted as described above. The RNA samples were treated with TurboDNase (Invitrogen, Germany) to ensure the complete removal of genomic DNA. We then purified the RNA using the NucleoSpin RNA extraction kit. The High-Capacity RNA to cDNA kit (Applied Biosystems) was used to generate cDNA according to the manufacturer’s recommendations. Gene-specific primers, designed using PrimerQuest (Integrated DNA Technologies, Coralville, IA, USA); PrimerQuest">http://eu.idtdna.com/PrimerQuest ) and purchased from Sigma-Aldrich, were used in a 10-µl reaction to quantify the p300/CBP mRNA, comprising 10 µM specific primers, 5 µL 2x Power SYBR Green PCR Master Mix and 2 µL cDNA template (50 ng cDNA per reaction mixture). The StepOnePlus Real-Time PCR System (Applied Biosystems) was used with a primary denaturation step at 95 °C for 5 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. We used three replicates for statistical analysis of target gene expression with REST2009 software [50 (link)]. The data were normalized against the ribosomal protein L32 (rpl32) gene in aphids. The sequences of all primers are provided in Table S1 .
6
RT-qPCR Validation with SsoAdvanced Supermix
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SsoAdvanced Universal SYBR Green Supermix from Bio-Rad Laboratories was used for RT-qPCR and performed following manufacturer’s protocol. All RT-qPCR plates were run on a CFX96 touch real-time PCR detection system (Bio-Rad, CA, USA). Primers utilized for RT-qPCR validations were designed using either QuantPrime69 or PrimerQuest from Integrated DNA Technologies (PrimerQuest">http://eu.idtdna.com/PrimerQuest ). The efficiency of each primer pair was tested before use by performing a standard curve, and the efficiency criteria for using a primer pair was 90% < E < 110%, with an R2 cut-off > 0.990. Housekeeping genes, ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (Atp5b), Calnexin (Canx) and Ribosomal Protein L13a (Rpl13a) were used for normalization of brain tissue RT-qPCR. Changes in gene expression were calculated using the CFX manager software program (Bio-Rad, CA, USA), using the ΔΔCt method with a fold change cut-off at ≥1.5 and p < 0.05 considered significant. All samples were run in triplicate and relevant positive and negative controls were run on each plate.
7
RT-qPCR Validation of Gene Expression
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Validations were carried out using RT-qPCR on inguinal WAT and iBAT from 12-week R6/2 mice and WT littermates. Further validations were then performed on 12- and 18-month Q175 mice and WT littermates. For RT-qPCR experiments, all samples were run in triplicate for each target gene and housekeeping gene, and relevant negative and positive controls were run on each plate. Melt curves were inspected for all assays, with the Tm checked to be within known specifications for each assay. Sample assay data points were included in data analysis only if detected with Ct < 37 and at least 3 Ct values lower than the corresponding negative control [27 (link)]. Any data that did not pass these criteria were omitted from all further analyses. Primers utilised for RT-qPCR validations (Table 1 ) were designed using QuantPrime [28 (link)] or PrimerQuest from Integrated DNA Technologies (PrimerQuest">http://eu.idtdna.com/PrimerQuest ).
8
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with the RNA exaction Kit (Qiagen) and reverse transcribed using random hexamer primers (Life Technologies). The cDNA was amplified following the procedure for quantitative real-time PCR analysis as described previously (17 (link)). The PCR primers were designed with Integrated DNA Technologies’ Primer Quest, and their sequences listed in Supplemental Table S1 . All reactions were performed in triplicates to ensure reproducibility. The PCR signals were normalized to those of GAPDH and displayed as relative folds of induction.
9
RT-qPCR Gene Expression Analysis
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First strand cDNA was synthesized from 3 μg total RNA using the Maxima First Strand cDNA Synthsis Kit (Thermo Fisher Scientific) according to the manufacture’s specifications. Primers and probes (Table S5) for RT-qPCR were designed using PrimerQuest (Integrated DNA Technologies). The RT-qPCR reaction mixture contains 1X Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific), 500 nM primers, 250 nm probe and 10 ng cDNA template. The pheS and alaS housekeeping genes were used as the references. For each condition, three biologically independent samples were used and the reactions were carried out in triplicate on an Mx3005P qPCR system (Agilent). Cycling conditions for all amplifications were one cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. For the RT-qPCR data, an average cycle threshold (CT) value was calculated from the triplicate reactions. Amplification efficiency and statistical analysis were performed using REST 200967 (link).
10
Total RNA Extraction and Quantification
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Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A total of 500ng of RNA was used as the template for the first-strand cDNA synthesis using ReverTraAceqPCR RT Kit (TOYOBO, Osaka, Japan) in accordance with the manufacturer’s protocol. The transcripts were quantified using Light Cycler 480Real-Time PCR system (Roche, Basel, Switzerland). Primers were designed using Primer Quest (Integrated DNA Technologies, Inc).
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