Droplet digital PCR (Bio-Rad, Hercules, CA) with DNA detection assays (Thermo Fisher) for sjKREC, sjTREC (both custom made, sequences available on request) and TaqMan Copy Number Reference Assay for human RNase P was performed using 250 ng of restriction digested (EcoRI, New England Biolabs) genomic DNA according to the manufacturer’s instructions. Relative quantity of sjKREC and sjTREC per (RNaseP / 2) ∗ 100,000 cells was measured with QuantaSoft v1.4 (Bio-Rad). Correlation among n = 12 duplicate measurements was 0.81 for sjKREC and 0.92 for sjTREC. sjKREC is correlated with B cells (r2 = 0.33), in particular transitional (r2 = 0.14) and naive (r2 = 0.22) B cells, whereas sjTREC is correlated with RTE CD4+ (r2 = 0.15) and CD8+ (r2 = 0.13) T cells.
Quantasoft v 1
Manufactured by Bio-Rad
Sourced in United States
QuantaSoft v.1.6 is a software application for digital PCR data analysis and reporting. It provides tools for the acquisition, analysis, and management of data from Bio-Rad's digital PCR instruments.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet reader, by Bio-Rad (19 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (16 mentions)
Qx100 droplet reader, by Bio-Rad (11 mentions)
Qx200 droplet generator, by Bio-Rad (10 mentions)
Ddpcr supermix for probe, by Bio-Rad (9 mentions)
Qx200 ddpcr system, by Bio-Rad (6 mentions)
C1000 touch thermal cycler, by Bio-Rad (6 mentions)
T100 thermal cycler, by Bio-Rad (5 mentions)
Super mix taqman, by Bio-Rad (5 mentions)
Primepcr ddpcr mutation detection assay kit, by Bio-Rad (5 mentions)
Droplet digital pcr, by Bio-Rad (4 mentions)
Qx200 droplet digital pcr ddpcr system, by Bio-Rad (4 mentions)
Qx200 autodg droplet digital pcr system, by Bio-Rad (4 mentions)
Qx100 droplet digital pcr system, by Bio-Rad (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (3 mentions)
Taqman probe, by Thermo Fisher Scientific (3 mentions)
Primepcr ddpcr mutation detection assay, by Bio-Rad (3 mentions)
Qx200, by Bio-Rad (3 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (3 mentions)
86 protocols using quantasoft v 1
1
Droplet Digital PCR Quantification of sjKREC and sjTREC
2
Quantifying CD40 Splice Variants in PBMCs
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RNA was extracted from total PBMCs and reverse transcribed using a high-capacity cDNA reverse transcription kit (Thermo Fisher). Droplet digital PCR (Bio-Rad) was performed using 50 ng cDNA according to the manufacturer’s instructions with predesigned gene expression assays (Thermo Fisher) for total CD40 (Hs00374176_m1, spanning exon 1-2), CD40 without exon 6 (Hs01008251_m1, spanning exon5-7) and housekeeping gene POLR2A (Hs00172187_m1) or custom assays (sequence available upon request) for CD40 without exon 5 (spanning exon 4-6) and without exon 5 and 6 (spanning exon 4-7). Relative quantity of the CD40 splice forms versus POLR2A and of the alternative splice forms versus total CD40 was measured with QuantaSoft™ v1.4 (Bio-Rad).
3
Quantifying STXBP1 Copy Number Using ddPCR
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We applied droplet digital PCR (ddPCR) technology to validate CNVs and to quantify the exact copy number. Copy number estimation of STXBP1 was performed using the QX200 ddPCR system (Bio-Rad Laboratories, Inc., Hercules, CA) using the TaqMan copy number probe Hs00269332_cn (Life Technologies, Carlsbad, CA). Prior to the copy number experiment, 250 ng of genomic DNA was digested with 4U of BtgI in a 10-μL reaction buffer (New England Biolabs, Ipswich, MA), 1 hour × 37°C incubation, and no enzyme denaturation. The 20-μL copy number reaction mix consisted of 10 μL of 2× ddPCR supermix for probes (Bio-Rad Laboratories), 1 μL of the copy number target assay (labeled with FAM), 1μL of the copy number reference assay (RNAseP, Life Technologies, part 4403326, labeled with VIC), 6 μL of water, and 2 μL of 25 ng/μL digested genomic DNA. The copy number assay was validated by temperature gradient to ensure optimal separation of target and RNAseP-containing droplets. Cycling conditions for the reaction were 95°C for 10 minutes, followed by 45 cycles of 94°C for 30 seconds and 60°C for 1 minute, 98°C for 10 minutes and finally a 4°C hold on a Life Technologies Veriti thermal cycler. Data were analyzed with QuantaSoft v1.4 (Bio-Rad Laboratories). Two reference DNA samples (NA10843 and HuRef) plus 3 nontemplate controls were included with the study samples.
4
TERT Expression Quantification in Breast Tumor FFPE Samples
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RNA extraction was performed for those patients’ samples in which tumor tissue from the same FFPE block was available. Total RNA was isolated using the RNeasy FFPE Kit (Qiagen, 73,504), following the manufacturer’s recommendations and then was reverse transcribed using SuperScript IV Reverse Transcriptase, following the manufacturer’s instructions (Invitrogen, 8,090,010). hTERT gene expression estimation was performed using the Droplet Digital PCR (ddPCR) technology, which has been proven to provide more precise and reproducible results in FFPE samples [73 (link)].
The QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad Laboratories, CA, USA) and Taqman probes (Life Technologies, USA), TERT probe Hs00972650_m1 and TBP probe Hs00427621_m1, as an endogenous control, were used in a duplex reaction mode. Different controls (no template, no reverse transcriptase (RT), and Human Universal RNA) were run in parallel with the study samples and the data was analyzed using Quanta-Soft v1.4 (Bio-Rad Laboratories). The TERT/TBP ratio of clinical breast FFPE samples was determined for each sample [74 (link), 75 (link)]. Then, the obtained ratios were calibrated for HeLa cell line transcript ratios. The results obtained represent the expression of breast tumor samples relative to HeLa cell line [22 (link)].
The QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad Laboratories, CA, USA) and Taqman probes (Life Technologies, USA), TERT probe Hs00972650_m1 and TBP probe Hs00427621_m1, as an endogenous control, were used in a duplex reaction mode. Different controls (no template, no reverse transcriptase (RT), and Human Universal RNA) were run in parallel with the study samples and the data was analyzed using Quanta-Soft v1.4 (Bio-Rad Laboratories). The TERT/TBP ratio of clinical breast FFPE samples was determined for each sample [74 (link), 75 (link)]. Then, the obtained ratios were calibrated for HeLa cell line transcript ratios. The results obtained represent the expression of breast tumor samples relative to HeLa cell line [22 (link)].
5
BAFF Gene Expression Quantification
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RNA was extracted from total PBMCs and reverse transcribed using a high-capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA). Droplet digital PCR (Bio-Rad, Hercules, CA) with gene expression assays (Life Technologies) for the BAFF gene TNFSF13B (Hs00198106_m1) and housekeeping gene POLR2A (Hs00172187_m1) was performed using 4 ng/μL cDNA. Relative quantity of TNFSF13B vs POLR2A was measured with QuantaSoft v1.4 (Bio-Rad). Correlation between separate experiments (n = 68) was 0.75.
6
Quantifying Immune Cell Markers via ddPCR
7
Quantifying Transgene Copy Number by ddPCR
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Droplet digital PCR (ddPCR) was performed to quantify NPTII (and thus transgene) copy number using a Biorad QX200 Droplet Digital PCR system (BioRad, Hercules, CA, USA). The concentration of NPTII (transgene), FIE (AT3G20740; endogenous single copy gene), and LYS (AT5G62150; endogenous single copy gene) was assessed using 2 ng of input genomic DNA. The rounded average between NPTII/FIE and NPTII/LYS ratios was finally used to determine the NPTII copy number. Droplet generation was performed according to the manufacturer’s protocol. Following droplet generation, the templates were amplified in a T100 thermal cycler (BioRad, Hercules, CA, USA). Fluorescence reads of the individual droplets were analyzed using Quanta Soft v1.7 (BioRad, Hercules, CA, USA). For each sample and probe, experiments were performed in technical duplicates. Primer and probe sequence information is shown in Additional file 1 : Table S14. Probes were custom designed and acquired from Life Technologies (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA).
8
Quantitative Gene Expression Analysis
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Total RNA was isolated using TRIzol reagent (Invitrogen) and then processed with ‐ RQ1 RNase‐Free DNase kit (Promega) to eliminate genomic DNA contamination. First‐strand cDNA was synthesized with a First Strand cDNA Synthesis Kit (Fermentas) using equal amount of RNA. Prior to digital droplet PCR, qRT‐PCR was used to determine the Cp value of mouse β‐Actin. Then qRT‐PCR normalized cDNA was amplified using 2× EvaGreen ddPCR Mastermix and thermal cycled at 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s and 58 °C for 1 min. Signal was then stabilized at 4 °C for 5 min followed by inactivation at 90 °C for 5 min. Droplets were then read by a QX200 Droplet Digital PCR System and analyzed with QuantaSoft V1.7 (BioRad).
9
Droplet Digital PCR for BRAF V600E Mutation Detection
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DNA was extracted on a Qiacube semiautomated robotic device (Qiagen, Valencia, CA) using either the QIAamp DNA Mini Kit (Qiagen) from 17 FNA washout samples and 6 frozen tissue samples, or the QIAamp DNA FFPE Tissue Kit (Qiagen) for paraffin-embedded tissue sections, according to the instructions of the manufacturer. BRAF T1799A (V600E) mutational analysis was performed using the PrimePCR ddPCR mutation detection assay (BIO-RAD, Hercules, CA) on a BIO-RAD QX200 droplet digital PCR (ddPCR) system. Each reaction included 10 μl of 2x ddPCR supermix for probes (no dUTP), 1 μl of BRAF V600E primer/probe mix (FAM), 1 μl of BRAF wild type primer/probe mix (HEX), and 40–100 ng of genomic DNA. The presence of mutation and the fractional abundance of the mutant allele was determined with QuantaSoft v.1.7 (BIO-RAD).
10
Droplet Digital PCR RNA Quantification
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Cellular RNA was extracted using RNA QuickExtract (Lucigen) without DNase. RNA was quantified using RiboGreen (Thermo Fisher) and normalized. Total RNA was reverse transcribed using iScript Advanced cDNA Synthesis Kit (BioRad) with 0.4 μM reverse primer for transcription. Reverse transcription product was amplified using 2x EvaGreen ddPCR Mastermix and thermal cycled at 95 °C for 3 min followed by 40 cycles of 95 °C for 30 s and 52.4 °C for 1 min. Signal was then stabilized at 4 °C for 5 min followed by inactivation at 90 °C for 5 min. Droplets were then read by a QX200 Droplet Digital PCR System and analyzed with QuantaSoft V1.7 (BioRad).
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