Unlabelled single-stranded cDNA was synthesized following the same procedure as for labelled cDNA but using a mixture of unlabelled dNTPs (10 mM each). Custom TaqMan® FAM-MGB Assay-by-Design primers and probes (Life Technologies) are provided in the S1 Table and amplification with TaqMan® Universal Master Mix 2x (Life Technologies) was run in a 7900HT Fast Real Time PCR system using SDS 3.1 software (Life Technologies) following the manufacturer’s instructions. The gGAPDH was the reference gene and the fold-change values were calculated with efficiency-corrected normalized quantities [22 (link)].
Taqman universal master mix 2x
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Universal Master Mix 2X is a ready-to-use solution for real-time PCR applications. It contains all the necessary components for efficient amplification and detection of target DNA sequences, including a DNA polymerase, dNTPs, and proprietary buffer system.
Automatically generated - may contain errors
Lab products found in correlation
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Fam nfq mgb probes, by Thermo Fisher Scientific (2 mentions)
Taqman assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quantstudio 7 flex system, by Thermo Fisher Scientific (1 mentions)
Hight capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid extraction kit, by Qiagen (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Taqman assay reagent, by Thermo Fisher Scientific (1 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Cfx96 touch real time pcr, by Bio-Rad (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rotor gene q real time pcr system, by Qiagen (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood midi kit, by Qiagen (1 mentions)
10 protocols using taqman universal master mix 2x
1
Quantitative Real-Time PCR Assay
2
Real-Time PCR Quantification of Gene Expression
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Unlabelled single stranded cDNA was synthesized following the same procedure described above but using a mixture stock of 10 mM of each dNTP. Custom TaqMan® MGB Assays-by-Design (specifically FAM-NFQ MGB probes) (Life Technologies) were run in a 7900HT Fast Real Time PCR system (Life Technologies) using TaqMan® Universal Master Mix 2x (Life Technologies) following the manufacturer’s instructions. Thermal cycling was as follows: 95°C for 5 min, 40 cycles [95°C for 30”, 60°C for 1 min]. PCR efficiencies were calculated by the standard curve best fit method from a triplicate dilution series experiment for each gene and cDNA sample (Pro-Pper/Amastigotes). Coefficients of variation were previously checked. Fold changes were calculated with respective efficiency-corrected normalized quantities in the same fashion as for microarray data. Normalized quantities were calculated by dividing the raw quantity value (efficiency to the power of –Ct) of the gene of interest by that of the endogenous control (GAPDH gene of L. infantum). Sequences of primers and probes are listed in the Additional file 1 .
3
Relative quantification of gene expression by qRT-PCR
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Unlabeled single stranded cDNA was synthesized following the same procedure as for microarray hybridization but using a mixture stock of 10 mM each dNTP. Custom TaqMan MGB Assay-by-Design (specifically primers and FAM-NFQ MGB probes, Life Technologies) were mixed with 1:5 serial dilutions of cDNA samples (10, 2 and 0.4 ng cDNA per reaction) and with TaqMan Universal Master Mix 2X (Life Technologies) in a final reaction volume of 10 μl. Primer and probe sequences are listed in S1 Table . The qRT-PCR reactions were run in a 7900HT Fast Real Time PCR system using the SDS 4.1. software (Life Technologies) following the procedure specified by the manufacturer. The thermal cycling conditions were: 95°C for 5 min; 40 x [95°C for 30”; 60°C for 1 min, data acquisition]. After checking coefficients of variation, PCR efficiencies were calculated by the standard curve best fit method using the data obtained in the triplicate dilution series experiment for each gene and cDNA sample (Pro-Pper/Pro-Stat). The normalized quantities were calculated by dividing the efficiency-corrected raw quantities (efficiency to the power of–Ct) for the gene of interest by those for the reference gene (L. infantum gGAPDH). Fold changes were obtained by dividing the normalized quantities (Pro-Pper/Pro-Stat). This procedure is based on specifications provided by Bookout et al. [35 ].
4
Quantitative PCR Gene Expression Analysis
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Total RNA was extracted using Qiagen RNeasy Lipid extraction kit following manufacturer’s instructions. Total RNA was treated with DNase I (Qiagen) at 37 °C for 30 min, followed by inactivation at 75 °C for 5 min. Reverse-transcription quantitative PCR assays were performed using an Applied Biosystems QuantStudio 7 Flex System. Total RNA (1 μg) was reverse transcribed with random hexamers using Hight-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, ThermoFisher Scientific) following the manufacturer’s protocol. Gene expression levels were determined with the Taqman Universal Master mix (2x) using Taqman assays (Applied Biosystems). The 18S transcript was used as an internal control to normalize the variations for RNA amounts. Gene expression levels are expressed relative to 18S mRNA levels. All primers used in this study were provided by Thermofisher.
5
Temporal Regulation of NOX mRNA
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Total RNA was extracted from CGN grown in depolarizing conditions from 0 to 5 DIV using Trizol Reagent according to the manufacturer's instructions. RNA quality was assessed by denaturing agarose gel electrophoresis and with NanoDro p2000 spectrophotometer (Thermo Scientific, USA). One µg of total RNA from each sample was reverse transcribed into cDNA using M-MVL Reverse Transcriptase with an oligo (dT)12-18 primer. One microgram of cDNA was used to determine the relative gene expression, which was performed in a thermal cycler Rotor-gene 6000 (Corbett Life Science), using TaqMan Universal Master Mix 2X and TaqMan Assay reagent for NOX1 (Rn00586652_m1), NOX2 (Rn00576710_m1), and GAPDH (Rn01775763_g1; Applied Biosystems®, USA). The relative level of amplified mRNA was normalized to the expression of the housekeeping gene GAPDH. The average Ct value of the endogenous control (GAPDH) for every sample was subtracted from the Ct value for each target gene, resulting in the ΔCT value. Fold change was calculated using the 2−ΔΔCT method where the comparative cycle threshold (ΔΔCT) was defined as the difference between ΔCT of 1 to 5 DIV minus ΔCT of the DIV in which the expression reached its maximum.
6
Genetic Variants in BLK and BANK1
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We used a TaqMan® SNP genotyping assay (Applied Biosystems, Foster City, CA) for the BLK rs13277113A/G and rs2736340T/C and BANK1 rs10516487C/T (R61H) and rs3733197G/A (A383T) genotypes. The Bio-Rad CFX Manager 3.1 software implemented in the CFX96 Touch TM Real-Time PCR of Bio-Rad (Bio-Rad, California, USA) was used to determinate the BLK and BANK1 genotypes in an allelic discrimination plot. The distribution of each BLK and BANK1 genotype was determined by two independent researchers. Additionally, 70% of all samples (including cases and controls) were genotyped twice with a reproducibility of 100%. PCR conditions for each amplicon and sample were as follows: 10 ng DNA per sample, 2.5 μl of TaqMan® Universal Master Mix (2X) (Applied Biosystems, Foster City, CA), 2.435 μl of nuclease-free water, and 0.065 μl of TaqMan probes (Applied Biosystems). The PCR protocol for amplification was as follows: pre-PCR (one cycle); at 50°C for 2 min and at 95°C for 8 min, followed by 45 cycles of denaturing at 95°C for 15 s and annealing an extension at 60°C for 1 min.
7
Quantification of Dopaminergic Neuron Transcripts
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About 100 SNpc neurons were LCM in triplicates from each tier from the same healthy controls used for the RNA‐seq analysis. The total RNA was extracted using PicoPureTM RNA Isolation Kit (Applied Biosystems, Foster City, CA) from 3 LCM preparations for each tier per case and pooled during elution phase. This step was carried out to gain at least ~50ng of RNA, which was then reverse transcribed using SuperScript ViLO cDNA synthesis kit (Invitrogen, Carlsbad, CA). The obtained cDNA was assayed on a Rotor‐Gene Q Real‐Time PCR System (QIAGEN) using TaqMan Gene Expression Assays (Life Technologies, Carlsbad, CA) by following standard protocols (Table1 ). Reactions were performed in duplicates using the FAM dye‐labeled assay. No‐template controls were run to determine any contamination. Amplification for TaqMan probes reactions was performed in a 20μl reaction volume, using 10μl TaqMan Universal Master Mix 2X (Applied Biosystems), 2μl cDNA, and 1μl TaqMan Probe 20x. The comparative delta Ct method (also known as the 2‐ΔΔCt method) was used to calculate the relative fold change in gene expression. Data were normalized to housekeeping genes (GAPDH and B2M) and gene expression in the ventral tier of SNpc (used as control = 1).
8
Molecular Diagnosis of Toxoplasmosis
9
Isolation and qPCR Analysis of VDR Gene
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The total RNA from PBMCs preserved with TRIzol (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was isolated. An amount of 200 µL of chloroform was added and centrifuged at 12,000× g for 15 min at 4 °C. Once the supernatant was removed, 500 µL of isopropanol was added and left on ice for 10 min. Additional centrifugation for 10 min at 12,000× g at 4 °C was performed. Once the pellet was isolated and washed in 75% ethanol, it was air-dried for 30 min. The purified RNA was dissolved in 30 μL of RNase-free water and subsequently was reverse transcribed into cDNA using High-Capacity Reverse Transcription kits (Thermo Fisher Scientific, Waltham, MA, USA). qPCR analysis was conducted using TaqMan probe (Hs01045843) (Thermo Fisher Scientific, Waltham, MA, USA) and TaqMan Universal Master Mix 2X following the manufacturer’s instructions. The relative difference in VDR gene expression between OP and CTR subjects was calculated using 2−ΔΔCt normalized to GAPDH levels as the internal control.
10
Genotyping of CELSR2 rs629301
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Genomic DNA from all subjects were extracted from EDTA treated whole blood samples using standard commercial systems and stored at 4°C degrees.
Genotyping of rs629301 in CELSR2 gene was carried out by a validated commercial TaqMan assay (Thermo Fisher) in a ViiA7 Real Time PCR System (Thermo Fisher). Each assay reaction was composed of 5 μL TaqMan Universal Master Mix (2X) (Thermo Fisher), 0.25 μL assay mix (40X), 1-2 μL DNA (total input of 1-10 ng template), 3.75-2.75 μL H2O. Thermal cycling was performed at the following conditions: 95°C, 10 minutes; (95°C, 15 seconds; 60°C, 1 minute) for 40 cycles. The genotyping call was done with SDS software v.1.3.0 (ViiA7 software RUO, Life Technologies).
Genotyping of rs629301 in CELSR2 gene was carried out by a validated commercial TaqMan assay (Thermo Fisher) in a ViiA7 Real Time PCR System (Thermo Fisher). Each assay reaction was composed of 5 μL TaqMan Universal Master Mix (2X) (Thermo Fisher), 0.25 μL assay mix (40X), 1-2 μL DNA (total input of 1-10 ng template), 3.75-2.75 μL H2O. Thermal cycling was performed at the following conditions: 95°C, 10 minutes; (95°C, 15 seconds; 60°C, 1 minute) for 40 cycles. The genotyping call was done with SDS software v.1.3.0 (ViiA7 software RUO, Life Technologies).
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