Total cell-associated SIV RNA was quantified using RT-ddPCR using 100 ng of RNA, a 1-Step RT-ddPCR Advanced kit for probes (Bio-Rad; cat. no. 1864022), and the same set of primers and probe used for SIV plasma viral load quantification. ddPCR was carried out on a Bio-Rad QX200 AutoDG digital droplet PCR system. In brief, 22 μl of reaction mix was used for droplet generation using a QX200 droplet generator, the ddPCR plate having the emulsified samples was heat sealed with foil (Bio-Rad; cat. no. 181-4040), and amplification occurred in a C1000 Touch thermal cycler (Bio-Rad, CA, USA). After thermal cycling, ddPCR plates were transferred to the QX200 droplet reader (Bio-Rad) for droplet count and fluorescence measurement. Positive droplets with amplified products were separated from negative droplets without target amplicon by applying a fluorescence amplitude threshold, and the absolute quantity of RNA per sample (copies/µl) was determined using QuantaSoft software. For quantification of cell-associated SIV DNA, the same methodology was used as described above without using the reverse transcription step and with 2× ddPCR Supermix for probes (no dUTP) (Bio-Rad; cat. no. 1863024) instead of the 1-Step RT-ddPCR Advanced kit.
1 step rt ddpcr advanced kit for probes
Manufactured by Bio-Rad
Sourced in United States
The 1-Step RT-ddPCR Advanced Kit for Probes is a laboratory product designed for the detection and quantification of RNA targets using the droplet digital PCR (ddPCR) technology. The kit provides the necessary reagents and components to perform a one-step reverse transcription and digital PCR reaction in a single tube.
Automatically generated - may contain errors
Lab products found in correlation
Qx200 droplet digital pcr system, by Bio-Rad (5 mentions)
Qx200 droplet reader, by Bio-Rad (2 mentions)
Qx200 droplet generator, by Bio-Rad (2 mentions)
Biosprint 96 platform, by Qiagen (2 mentions)
Nucleomag vet kit, by Macherey-Nagel (2 mentions)
Ddpcr supermix for probe, by Bio-Rad (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Qx200 autodg, by Bio-Rad (1 mentions)
Biorad real time cfx96 detection system, by Bio-Rad (1 mentions)
Cfx96 real time pcr cycler, by Bio-Rad (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
C1000 touch thermocycler, by Bio-Rad (1 mentions)
Qx100 qx200 droplet generator, by Bio-Rad (1 mentions)
Qx100 qx200 droplet reader, by Bio-Rad (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Qx200 instrument, by Bio-Rad (1 mentions)
Droplet generation oil for probe, by Bio-Rad (1 mentions)
Quantasoft software, by Bio-Rad (1 mentions)
9 protocols using 1 step rt ddpcr advanced kit for probes
1
Quantification of Cell-Associated SIV RNA and DNA
2
SARS-CoV-2 Viral Load Quantification
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RNA from combined nasal/oral swabs and organ samples was extracted using the NucleoMag® VET Kit (Macherey-Nagel, Düren, Germany) in combination with a Biosprint 96 platform (Qiagen, Hilden, Germany). Each extracted sample was eluted in 100 µl. Viral RNA genome was detected and quantified by real-time RT-qPCR on a BioRad real-time CFX96 detection system (BioRad, Hercules, USA). Target sequence for amplification was the viral RNA-dependent RNA polymerase (WHO, https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2 ). Genome copies per µl RNA template were calculated based on a quantified standard RNA, where absolute quantification was done by the QX200 Droplet Digital PCR System in combination with the 1-Step RT-ddPCR Advanced Kit for Probes (BioRad, Hercules, USA). The limit of detection was calculated to be 10 copies per reaction.
3
Quantitative detection of avian H9N2 virus
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RT-qPCRs of organs, oral and rectal swabs in the first experiment (avian origin H9N2) were performed as described before [17 (link)].
In order to detect A/bat/Egypt/381OP/2017 (H9N2) viral RNA in the second experiment, a specific primer and probe system was designed (Table 1 ). For process control, a genomic nucleic acid was co-amplified in the PCR runs using the HEX channel [18 (link)]. The final composition of the RT-qPCR reactions was 1.75 μL of RNase-free water, 6.25 μL of 2x qScript XLT One-Step RT-qPCR ToughMix (Quanta, Beverly, MA, USA), 1 μL of primer-probe-mix-FAM, 1 μL of beta-actin DNA-mix2-HEX and 2.5 µL of template RNA. All RT-qPCRs were run on the CFX 96 real-time PCR cycler (Bio-Rad, Hercules, CA, USA). The temperature profile used was 10 min at 50 °C (reverse transcription), 1 min at 95 °C (inactivation of the reverse transcriptase/activation Taq polymerase) followed by 45 cycles of 10 s at 95 °C (denaturation), 30 s at 57 °C (annealing) and 30 s at 68 °C (elongation). Fluorescence values (FAM, HEX) were collected during the annealing step. Absolute quantification was done using a standard of known concentrations, corresponding to the RNA of the original virus used for inoculation. Quantification was established by the QX200 Droplet Digital PCR System in combination with the 1-Step RT-ddPCR Advanced Kit for Probes (BioRad, Hercules, CA, USA).
In order to detect A/bat/Egypt/381OP/2017 (H9N2) viral RNA in the second experiment, a specific primer and probe system was designed (
4
SARS-CoV-2 N1 Nucleocapsid ddPCR Protocol
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The N1 nucleocapsid primers from the CDC assay were used for all ddPCR data with the 1-step RT-ddPCR Advanced Kit for Probes (Bio-Rad Catalog # 1864022). In brief, a 20 μL reaction comprising 2 μL reverse transcriptase, 1 μL DTT, 1.5 μL primer master mix, 5.5 μL water, and 5 μL input RNA (or crude lysate) was used for each sample. Droplets were generated with the QX200 Droplet Digital PCR System (Bio-Rad), sealed, and reverse transcription was performed by incubating at 50 °C for 60 min followed by 95 °C for 10 min per manufacturer’s protocol. The reaction was then thermocycled with the same conditions as used for the CDC EUA-approved bulk qRT-PCR assay described above, and then read with the QX200 Droplet Reader (Bio-Rad) with thresholds between positive and negative drops set by QuantaSoft Software and confirmed by manual inspection. For qualitative assessment of fluorescence, thermocycled drops were imaged using the EVOS Cell Imaging System (Thermo Fisher).
5
Quantifying Circulating Oncogenic Transcripts
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Two transcripts, POU6F2-AS2 and AC022126.1, both of which had no measurable transcripts in the healthy donors in the cfRNA-seq data, were further analyzed. One of these two transcripts, POU6F2-AS2 has previously been associated with cancer pathogenesis. Circulating levels of POU6F2-AS2 and AC022126.1 transcripts were measured by means of RT-ddPCR using the 1-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, Hercules, CA, USA) in samples from our validation cohort, which included 45 stage IV lung cancer patients, 39 stage I–III lung cancer patients, 20 PDAC, 22 bladder cancer, and more than 65 healthy control samples. Samples from the validation cohort were both plasma and serum. All reactions were performed in duplicates using 2 µL of cfRNA from each sample. The reaction components were constituted following manufacturers instruction to a final volume of 22 µL, of which 20 µL were used for droplet generation in a QX100™/QX200™ droplet generator (Bio-Rad). RT-ddPCR reactions were performed in a C1000 Touch™ thermocycler (Bio-rad), and droplets were read in a QX100™/QX200™ droplet reader (Bio-Rad). The raw transcript concentration (copies/20 µL reaction) was used to determine the absolute transcript load per ml of plasma/serum using the formula:
where
For tissue-derived samples, RT-ddPCR was performed on 4 ng of total RNA for each sample and data expressed per ng of RNA.
where
For tissue-derived samples, RT-ddPCR was performed on 4 ng of total RNA for each sample and data expressed per ng of RNA.
6
Quantifying Bat H9N2 Viral RNA
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Animal organ samples of about 0.1 cm3 size were first homogenized in a 2 mL Eppendorf-tube containing 1 mL of Hank’s balanced salts MEM and Earle’s balanced salts MEM (2 mM L-glutamine, 850 mg L−1 NaHCO3, 120 mg L−1 sodium pyruvate, and 1% penicillin–streptomycin) at 300 Hz using a Tissuelyser II (Qiagen, Hilden, Germany). From each homogenized organ, swab or nasal wash sample, 100 µl was extracted via the NucleoMag Vet kit (Macherey&Nagel, Düren, Germany) according to the manufacturer’s instructions on a Biosprint 96 platform (Qiagen). Viral RNA was detected by RT-qPCR using bat H9N2-specific primers and probes38 (link). Absolute quantification was done using a standard of known concentrations, corresponding to the RNA of the original virus used for inoculation. Quantification was established by the QX200 Droplet Digital PCR System in combination with the 1-Step RT-ddPCR Advanced Kit for Probes (BioRad, Hercules, CA, US). Viral titers of ferret nasal washing samples were determined by TCID50 endpoint dilution assay on MDCKII cells.
7
Circulating MORF4L2 Transcript as NSCLC Biomarker
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We previously reported on the utility of cfRNA for disease detection in solid tumors.12 Based on our data (cfRNA‐seq) and analysis of publicly available the cancer genome atlas and gene expression omnibus (GEO) data sets from NSCLC tumors tissues, we identified the most relevant of the differentially abundant protein‐coding transcripts for further validation. To this end, all differentially abundant cfRNA transcripts (in both plasma and tumor tissues) were first subjected to feature selection using the Boruta package in R. The most important transcripts were then classified according to their expression levels in NSCLC tumor tissue. We selected the most abundant protein‐coding cfRNA transcript, MORF4L2 for further investigation. Circulating levels of MORF4L2 transcripts were measured by means of RT‐ddPCR using the 1‐Step RT‐ddPCR Advanced Kit for Probes (Bio‐Rad). All reactions were performed in 20 μl duplicate reaction using 2 μl of cfRNA from each sample. The QX100 ddPCR system was used for ddPCR experiments. The average raw MORF4L2 cfRNA transcript concentration (copies/20 μl reaction) obtained from duplicate reactions, was used to determine the absolute transcript concentration expressed in copies per ml of plasma.
8
Synthetic SARS-CoV-2 RNA Controls Characterization
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Three synthetic SARS-CoV2 genomic RNA products were purchased from Twist
Bioscience for the ILC2 study: B.1 (Twist Control 10), Alpha (B.1.1.7; Twist
Control 15), and Beta (B.1.351; Twist Control 16). These single-stranded RNA
controls are manufactured by in vitro transcription from 6 non-overlapping 5-kb
synthetic gene fragments. According to the manufacturer, the synthetic RNAs
cover 99.9% of the bases of the viral genomes that were predominant in the
United States, including the U.S.B.1, United Kingdom (Alpha [B.1.1.7]), and
South Africa (Beta [B.1.351]) variants, with GISAID names: USA/CA-PC101P/2020,
England/205041766/2020, and South Africa/KRISP-EC-K005299/2020, respectively.
Droplet digital RT-PCR (RT-ddPCR)-based quantification of these controls was
performed by the Cornell University Genomics Facility (QX200 instrument;
Bio-Rad). The CDC N1 primers and probe (IDT) were used for this analysis with
the 1-step RT-ddPCR advanced kit for probes (Bio-Rad), on duplicate serial
dilutions of the templates. The concentrations of the original Twist B.1, Alpha
(B.1.1.7), and Beta (B.1.351) controls were determined by RT-ddPCR as 150,000,
345,000, and 300,000 copies/μL, respectively. Serial dilutions of the controls
were then made in nucleic acid dilution solution from the VetMAX Xeno internal
positive control DNA kit (Applied Biosystems) to levels of 2 × 105 to
2 copies/μL in 10-fold dilutions.
Bioscience for the ILC2 study: B.1 (Twist Control 10), Alpha (B.1.1.7; Twist
Control 15), and Beta (B.1.351; Twist Control 16). These single-stranded RNA
controls are manufactured by in vitro transcription from 6 non-overlapping 5-kb
synthetic gene fragments. According to the manufacturer, the synthetic RNAs
cover 99.9% of the bases of the viral genomes that were predominant in the
United States, including the U.S.B.1, United Kingdom (Alpha [B.1.1.7]), and
South Africa (Beta [B.1.351]) variants, with GISAID names: USA/CA-PC101P/2020,
England/205041766/2020, and South Africa/KRISP-EC-K005299/2020, respectively.
Droplet digital RT-PCR (RT-ddPCR)-based quantification of these controls was
performed by the Cornell University Genomics Facility (QX200 instrument;
Bio-Rad). The CDC N1 primers and probe (IDT) were used for this analysis with
the 1-step RT-ddPCR advanced kit for probes (Bio-Rad), on duplicate serial
dilutions of the templates. The concentrations of the original Twist B.1, Alpha
(B.1.1.7), and Beta (B.1.351) controls were determined by RT-ddPCR as 150,000,
345,000, and 300,000 copies/μL, respectively. Serial dilutions of the controls
were then made in nucleic acid dilution solution from the VetMAX Xeno internal
positive control DNA kit (Applied Biosystems) to levels of 2 × 105 to
2 copies/μL in 10-fold dilutions.
9
Droplet Digital RT-PCR for Virus Detection
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