Equal amounts of RNA were reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR Kit (Thermo Fisher Scientific) according to the manufacturer’s protocols. The expression levels of the different zebrafish genes analyzed in this study were measured by RT-PCR or RT-qPCR using the primers listed in Supplementary Table 1 and the TaqMan assays reported in Supplementary Table 4 , respectively. Zebrafish beta-actin (actb1) was used as endogenous control in all reactions (McCurley and Callard, 2008 (link)). Band intensities from RT-PCR reactions were manually quantified by drawing boxes of the same area using the Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). RT-qPCR reactions were performed according to the manufacturer’s protocol using ddPCR SuperMix for Probes (no dUTP, Bio-Rad Laboratories 1863024) and a QX200 Droplet Digital PCR System (Bio-Rad Laboratories). Results were analyzed with the Quanta Soft software 1.7.4.0917 (Bio-Rad Laboratories) and are presented as absolute quantification values (normalized to actb1).
Quantasoft software 1
Manufactured by Bio-Rad
Sourced in Germany
QuantaSoft software 1.7 is a digital PCR analysis software. It is designed to analyze digital PCR data and generate quantitative results.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet reader, by Bio-Rad (3 mentions)
Qx100 droplet generator, by Bio-Rad (3 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (2 mentions)
Ddpcr supermix for probes no dutp, by Bio-Rad (2 mentions)
Ddpcr master mix for probes, by Bio-Rad (2 mentions)
Ddpcr supermix for probe, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis supermix for qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Vic labeled rpp30 rnase p assay, by Thermo Fisher Scientific (1 mentions)
Qx100 droplet reader, by Bio-Rad (1 mentions)
C1000 touch, by Bio-Rad (1 mentions)
Thermal cycler, by Thermo Fisher Scientific (1 mentions)
Qx200 system, by Bio-Rad (1 mentions)
6 protocols using quantasoft software 1
1
Quantitative Gene Expression Analysis in Zebrafish
2
Copy Number Variation Analysis in Germline and Corticotropinoma Samples
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CNV analysis was performed in 116 germline and 29 corticotropinoma DNA samples using FAM-labeled assays binding CABLES1 exon 1, intron 3–exon 4 and exon 10 (TaqMan CNV assays Hs07536236_cn, Hs02003953_cn, and Hs00413958_cn, respectively, Thermo Fisher Scientific) a VIC-labeled RPP30 (Rnase P) assay (Thermo Fisher Scientific 4403326) as an internal control, and the ddPCR SuperMix for Probes (no dUTP) (Bio-Rad 1863024) in a QX200 Droplet Digital PCR System (Bio-Rad). Results were analyzed with the Quanta Soft software 1.7.4.0917 (Bio-Rad).
3
Quantification of Vector Copy Number
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Quantitation of VCN was performed via ddPCR (Bio-Rad, CA, USA). Primers and probes were designed by Bio-Rad and the unique assay numbers were dCNS219891929 (rhesus TERT gene as internal control, Fluorophore: HEX) and dCNS749431138 (HIV RRE gene found in both vectors, Fluorophore: FAM). Droplets were generated via the QX100 Droplet Generator (Bio-Rad) and assayed on the QX200 droplet reader (Bio-Rad) via a standard protocol.46 (link) Data were analyzed by QuantaSoft software 1.7 (Bio-Rad). RhCMV DNA copy numbers in plasma and saliva were determined via real-time qPCR as previously described.36 (link)
4
Absolute Quantification by ddPCR
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One ddPCR reaction contained 10 μl of ddPCR Master Mix for Probes (Bio-Rad, Munich, Germany), 12.5 pmol of each primer, 6.25 pmol of each probe, and 5 μl of template DNA. Samples were prepared in duplicate with 10 % additional volume and droplets generated (QX100 droplet generator, Bio-Rad, Munich, Germany). PCR was performed as following: initial denaturation at 95 °C for 10 min, amplification in 40 cycles at 95 °C for 30 s and 60 °C for 1 min, and enzyme deactivation at 98 °C for 10 min. For all steps, a ramp time of 2 °C/s was used. Afterwards, the droplets were analyzed in the QX100 droplet reader (Bio-Rad, Munich, Germany). The data were analyzed with the Quantasoft software 1.7 (Bio-Rad, Munich, Germany).
5
Droplet Digital PCR Protocol for DNA Detection
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Unless otherwise stated, one ddPCR reaction contained 10 μl of ddPCR Master Mix for Probes (Bio-Rad, Munich, Germany), 12.5 pmol of each primer (Table 1 ), 6.25 pmol of each probe (Table 1 ), and 5 μl of template DNA. Samples were prepared in duplicate with 10% additional volume. 20 μl sample and 70 μl reader oil were transferred to respective wells in the cartridges and attached with gaskets. Droplets were generated in the QX100 droplet generator (Bio-Rad, Munich, Germany) and transferred (≈ 40 μl) to a 96-well plate and heat-sealed. Unused wells in the cartridge were filled with 10 μl of ddPCR Master Mix for Probes mixed with 10 μl H2O. PCR was performed as follows: initial denaturation at 95°C for 10 minutes, amplification over 40 cycles at 95°C for 30 seconds and 60°C for 1 minute and enzyme deactivation at 98°C for 10 minutes. For all steps a ramp rate of 2°C/s was used (Figs 1 and 2 : T100 (Bio-Rad, Munich, Germany), Figs 3 –6 : C1000 touch (Bio-Rad, Munich, Germany)). Afterwards, the droplets were analyzed in the QX200 droplet reader (Bio-Rad, Munich, Germany). The data were analyzed with Quantasoft software 1.7 (Bio-Rad, Munich, Germany).
6
Quantification of mtDNA Heteroplasmy
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The 3243A > G heteroplasmy of the 2SA, 2SD and 2KD cells were analyzed by the Droplet Digital PCR assay. The ddPCR mixture consisted of total DNA, 2× ddPCR Supermix for Probes (no dUTP) (Bio-Rad, Hercules, CA, 1863024), wild-type and mutant allele-specific probes, primer mixtures for the target gene, and the 2U/reaction Alu I enzyme (New England Biolabs, Ipswich, MA, R01375). All primers and probes were obtained from Bio-Rad. Sequences and other information are available at www.bio-rad.com with assay ID dHsaMDS556387941. Droplets were generated with the Bio-Rad QX200 system (Bio-Rad), in accordance with the manufacturer's instructions. The reactions were then subjected to PCR analysis with Thermal Cycler (Applied Biosystems, Waltham, MA). The number of fluorescence-positive droplets was analyzed using QX200 Droplet Reader (Bio-Rad). A two-color detection system (set to detect FAM and HEX) was used to analyze each droplet separately. The fluorescent droplets were counted and absolute quantification of the copy number of wild type and mutant at m.3243 was performed using QuantaSoft software 1.7 (Bio-Rad). The 3243A > G heteroplasmy was calculated as follows.
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