RNA was transcribed into cDNA using iScript Reverse Transcription Supermix (Bio-Rad, USA). cDNA quantification of MBP, MOG, and PLP in hOPCs was done with specific TaqMan gene expression probes (UniGene: Hs00921945_m1, Hs01555268_m1, and Hs00166914_m1, respectively) using EvaGreen Digital PCR Supermix with Droplet Generation Oil for EvaGreen and the QX200 droplet digital PCR system (ddPCR; Bio-Rad Quanta SOFT Analysis Pro v.1.0.596 software, Hercules, CA, USA). The results were expressed as the number of gene copies per 20 symbol microliter of mixture reaction.
Quantasoft analysis pro
Manufactured by Bio-Rad
Sourced in United States
The QuantaSoft Analysis Pro is a software package developed by Bio-Rad for the analysis of quantitative PCR (qPCR) data. It provides users with tools to analyze and interpret qPCR results. The software supports a range of qPCR platforms and offers features for data management, statistical analysis, and report generation.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet reader, by Bio-Rad (9 mentions)
Quantasoft, by Bio-Rad (7 mentions)
C1000 touch thermal cycler, by Bio-Rad (5 mentions)
Qx200 ddpcr evagreen supermix, by Bio-Rad (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Direct zol rna microprep kit, by Zymo Research (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Qx200 autodg droplet digital pcr system, by Bio-Rad (3 mentions)
Agilent 2100 expert software, by Agilent Technologies (3 mentions)
Iscript cdna supermix, by Bio-Rad (3 mentions)
C100 touch thermal cycler, by Bio-Rad (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Ddpcr supermix for probe, by Bio-Rad (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (2 mentions)
Qx200 droplet digital system, by Bio-Rad (2 mentions)
Qx200 evagreen digital pcr supermix, by Bio-Rad (2 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (2 mentions)
Automated droplet generator, by Bio-Rad (2 mentions)
Qx200 droplet generator, by Bio-Rad (2 mentions)
31 protocols using quantasoft analysis pro
1
Quantification of Myelin Genes in hOPCs
2
Quantifying Mutant Col3a1 Transcript
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Total RNA were extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) from TA of Col3a1+/G182R mice and Col3a1+/+ mice, stored at -80°C, according to the manufacturer’s instructions. Total RNA were measured (NanoDrop One, Thermo Fisher, Waltham, MA), normalized to 10ng/μL and then reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The cDNA was used as template for the droplet digital PCR (ddPCR) analysis as follows: ddPCR reaction mixture (12.5μL ddPCR Supermix for Probes (No dUTP) (Bio-Rad, Hercules, CA), 1 μL specific primers of WT and mutated alleles (S1 Table ), 20 ng cDNA, and water up to 20 μL) was used for droplet generation. PrimePCR ddPCR Expression Probe Assay: Vim, Mouse (0.5X, Bio-Rad, Hercules, CA) was used as the internal control to normalize the expression of Col3a1. After droplet generation (QX100 Droplet Generator, Bio-Rad Laboratories, Hercules, CA), 40 μL of sample was manually transferred to a 96-well plate (Eppendorf, Hamburg, Germany). Amplification was performed (40 cycles of amplification, temperature of hybridization 61°C) and fluorescent intensity was measured in a QX100 Droplet Reader (Bio-Rad Laboratories, Hercules, CA) and the signal data was analysed using QuantaSoft Analysis Pro (1.0.596, Bio-Rad Laboratories, Hercules, CA).
3
Droplet Digital PCR Quantification of Lhx4 Expression
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Total RNA was separately extracted from three control and three Lhx4 null mice at adult with RNeasy Mini Kits (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. cDNA was synthesized from total RNA using iScript cDNA Synthesis Kit (Bio-Rad). ddPCR was performed by using QX200 Droplet Digital Systems (Bio-Rad). QX200 EvaGreen Digital PCR Supermix (Bio-Rad) was used for the ddPCR reaction. The analysis was performed on the QuantaSoft Analysis Pro (Bio-Rad). The housekeeping gene Actb was used for internal control.
4
Genetic Profiling of SMN Genes
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All variants, but not the SMN1/SMN2 exon 7 copy number, were analyzed by direct sequencing in the control group to obtain an allelic frequency in the population. SMN1, SMN2, and RPP30 concentrations were measured using a Bio-Rad QX200 Droplet Digital PCR system. QuantaSoft Analysis Pro (Bio-Rad) was used for droplet cluster classification and Poisson function applications to calculate absolute and relative SMN1, SMN2, and RPP30 copy numbers. Details of the PCR and ddPCR assays are available in Supplement (eMethods, links.lww.com/NXG/A529 ).
5
Droplet Digital PCR for Copy Number
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Copy numbers of w and Adh were determined using droplet-digital PCR using a QX200 instrument (Bio-Rad, Inc.). Genomic DNA from single adult male flies was extracted using using the Monarch Genomic DNA Purification Kit (New England Biolabs), using a protocol we developed (dx.doi.org/10.17504/protocols.io.bp2l694qklqe/v1 , Loehlin (2022) (link)). Testing suggested this procedure was more reliable for copy number determination than single-fly “squish” extractions (Gloor and Engels 1992 ), which are simpler to perform but often showed irregular copy-number calls.
For digital PCR, 2 µL of genomic DNA prep was fragmented by restriction digest in 20 µL reactions with EcoRV-HF and HinDIII-HF (New England Biolabs) for 2h. 4 µL of digest product was assayed in 20 µL PCR reactions using Bio-Rad ddPCR Supermix for Probes (no dUTP) using the manufacturer’s recommended procedure. Assays were duplex, comparing copy number of control gene RpL32 to w or Adh. Primers and probes are listed inFile S1 . ddPCR results were inspected in Bio-Rad QuantaSoft Analysis Pro. Droplets were manually segmented, applying the same threshold to all samples simultaneously. Data were plotted using R package ggplot2.
For digital PCR, 2 µL of genomic DNA prep was fragmented by restriction digest in 20 µL reactions with EcoRV-HF and HinDIII-HF (New England Biolabs) for 2h. 4 µL of digest product was assayed in 20 µL PCR reactions using Bio-Rad ddPCR Supermix for Probes (no dUTP) using the manufacturer’s recommended procedure. Assays were duplex, comparing copy number of control gene RpL32 to w or Adh. Primers and probes are listed in
6
Droplet Digital PCR for Copy Number
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Digital droplet PCR (ddPCR) was performed using a BioRad QX200 system (BioRad, UK). Primers were designed for two genes per chromosome of interest: NRG1 and ANGPT2 (chromosome 27), SHTN1 and ACTC1 (chromosome 1), with MCM6 (chromosome 18) as the reference (all individuals were diploid for this chromosome) (Supplementary Table S1 ). Copy number was determined in duplicate in reactions containing 50 ng DNA, using EvaGreen chemistry (final concentration: 100 nM each primer, 1 × ddPCR Supermix for EvaGreen). C1000 Touch Thermal Cycler performed ddPCR reactions as follows: 95 °C (5 min), 40 cycles of: 95 °C (30 s) and 58 °C (1 min), followed by 4 °C (5 min), and 90 °C (5 min). Droplets were analysed using Bio-Rad QX200 Droplet Reader and QuantaSoft Analysis Pro (v1.0.596; BioRad, https://www.bio-rad.com/en-uk/product/qx200-droplet-digital-pcr-system?ID=MPOQQE4VY ). The copy number of each product was manually calculated relative to MCM6.
7
Quantification of Viral Nucleic Acids in Wastewater
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Nucleic acids were quantified through one-step ddRT-PCR for SARS-CoV-2 (N1 and N2), BCoV, and PMMoV using the BioRad QX200 droplet digital PCR systems (BioRad, CA). Depending on the laboratory, between one and four replicates were run per sample. Positive and negative controls were included on each plate. Data was processed and exported using QuantaSoft and QuantaSoft Analysis Pro (BioRad, CA). The concentration per reaction was converted to copies per volume of wastewater using dimensional analysis. For AA, errors are standard deviations of three replicate wells. For all other POTWs, errors are the standard deviations as the “total error” from the instrument, which includes errors associated with the Poisson distribution and variability among replicate wells.
8
Droplet Digital PCR Genotyping
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Confirmatory genotyping and allelic expression data were generated using the droplet digital PCR system (BioRad, Hercules, CA, USA). A 20 μL ddPCR reaction mixture containing custom-designed normal and mutant primer/probe sets (see Supplementary Table S2 ) was prepared according to manufacturer's directions, droplets generated, and nanoreactions cycled on a thermal cycler (C1000 Touch; Bio-Rad Laboratories). The average number of allelic transcripts per biological replicate (n = 3) was then determined with a droplet reader and analytical software (QX 200 with QuantaSoft Analysis Pro; Bio-Rad Laboratories).
9
Droplet Digital PCR for MTC Detection
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The QX200 Droplet Reader (Bio-Rad, USA) was used to read and count the fluorescent positive and negative droplets. Then, the data were analyzed using the QuantaSoft™ Analysis Pro software (version 1.0.596) (Bio-Rad, USA). Data from samples with 12,000 – 16,000 droplets were used for concentration calculations. Samples with a low number of droplets (< 10,000) were excluded from the analysis. According with the results for LOB, those samples with fewer than two positive droplets were considered “MTC-negative”, in contrast, samples were considered as “MTC-positive” when more than two droplets were found (Whale et al., 2020 (link)). Thus, the cut-off value of ddPCR-IS6110 assay was set at 3 positive droplets in 20 μL reaction mixture.
10
Quantitative Analysis of mtDNA Variants
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This was described in detail previously (Klein Gunnewiek et al., 2020 ). In short, ddPCR was performed on DNA extracted from neurons at DIV44, using ddPCR primers custom synthesized to amplify the mitochondrial MT-TL1 m.3243 region. Data analysis was performed with QuantaSoft Analysis Pro version 1.0.596 (Bio-Rad).
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