Peripheral blood from patients was collected in cell-free DNA collection tubes (Roche) at day one of the first administration of the systemic chemotherapy regimen as well as 4–6 weeks after the first blood sample. Blood samples were proceeded within 12 hours of collection via a 2-step centrifugation protocol. First, plasma was separated from the other blood components by centrifugation at 2000 x g for 20 minutes. After transferring the upper plasma layer to a new conical tube, it was respun at 3200 x g for 30 minutes to remove cell debris. Subsequently the resulting plasma supernatant was stored at −20 °C in 10 mL cryotubes (VWR) until DNA isolation. Circulating DNA isolation from 5–10 mL plasma was performed on the Chemagic 360 Instrument (Perkin Elmer) with the isolation kit CMG-1111 (chemagic cfDNA 10k Kit special H12) according to manufacturer’s instruction. Cell-free DNA was eluted in ~40 µL elution buffer. DNA quantification was performed with Qubit® dsDNA HS Assay Kit (Invitrogen) according to the instructions provided by the manufacturer and purity was determined by Agilent 2200 TapeStation System. Cell-free DNA was stored at −20 °C until further analysis.
Chemagic 360 instrument
Manufactured by PerkinElmer
Sourced in United States
The Chemagic 360 instrument is a fully automated nucleic acid extraction system designed for high-throughput processing of biological samples. The core function of the Chemagic 360 is to perform efficient and reliable extraction of DNA, RNA, and other biomolecules from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cobas 6800 system, by Roche (2 mentions)
Cryotubes, by Avantor (1 mentions)
Cell free dna collection tube, by Roche (1 mentions)
Agilent 2200 tapestation system, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Milli q water, by Merck Group (1 mentions)
Quantstudio 3 and 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Hap550k beadchip, by Illumina (1 mentions)
Axiom genome wide array plate system, by Thermo Fisher Scientific (1 mentions)
Vacutainer spray coated k2edta tubes, by BD (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Thermolabile proteinase k, by New England Biolabs (1 mentions)
Cfx96 touch real time pcr system, by Bio-Rad (1 mentions)
Picogreen reagent, by Thermo Fisher Scientific (1 mentions)
Enspire plate reader, by PerkinElmer (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
14 protocols using chemagic 360 instrument
1
Plasma-derived Cell-free DNA Collection
2
SARS-CoV-2 Inhibition by EGYVIR and Hydroxychloroquine
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The Vero-E6 cells (80–90% confluency) were infected at MOI of 0.1 of the virus then treated with EGYVIR and hydroxychloroquine as control at concentration 0.4 μg/ml and 2.2 μM, respectively. The cells were incubated in infection medium for 24, 48, and 72 h post infection at 37 °C in 5% CO2. Mock-infected cells without treatment were used as control. Cell culture supernatants were collected at each time point and virus was quantified by RT-qPCR.
Nucleic acid extraction for the cell culture isolates were done using the chemagic™ 360 instrument (Perkin Elmer, Waltham, Massachusetts, USA). Detection of SARS-CoV-2 RNA (ORF1 ab) was performed using Viasure Sars-CoV-2 Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain), the RT-PCR runs were done in triplicate and according to manufacturer’s recommendations. The obtained Ct values were changed to viral RNA copy numbers using a standard curve of ORF 1 ab assay, the viral inhibition in RNA copy number at each concentration was determined.
Nucleic acid extraction for the cell culture isolates were done using the chemagic™ 360 instrument (Perkin Elmer, Waltham, Massachusetts, USA). Detection of SARS-CoV-2 RNA (ORF1 ab) was performed using Viasure Sars-CoV-2 Real Time PCR Detection Kit (CerTest Biotec, Zaragoza, Spain), the RT-PCR runs were done in triplicate and according to manufacturer’s recommendations. The obtained Ct values were changed to viral RNA copy numbers using a standard curve of ORF 1 ab assay, the viral inhibition in RNA copy number at each concentration was determined.
3
Plasma DNA Extraction from Frozen Blood
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Blood was collected in three tubes and processed in three different ways. The first tube
was centrifuged within 24 hr of sampling and stored at −40°C until use. The second tube
was refrigerated at 4°C for 1 week after sampling and was then centrifuged and stored at
−40°C until use. The third tube was refrigerated at 4°C for 2 weeks after sampling and
then centrifuged and stored at −40°C until use. Frozen tubes were thawed by letting them
stand at room temperature (15–25°C) and then centrifuged to separate the hemolyzed blood
cells and plasma components. Plasma was collected from thawed tubes.
DNA was extracted from 1.5 ml plasma using a Custom NEXTprep cfDNA Auto
Kit (1.5 ml; PerkinElmer, Waltham, MA, USA) with a Chemagic 360
instrument (PerkinElmer). The extract was dissolved in Milli-Q water (Merck Millipore,
Burlington, MA, USA) to a final volume of 50 µl.
was centrifuged within 24 hr of sampling and stored at −40°C until use. The second tube
was refrigerated at 4°C for 1 week after sampling and was then centrifuged and stored at
−40°C until use. The third tube was refrigerated at 4°C for 2 weeks after sampling and
then centrifuged and stored at −40°C until use. Frozen tubes were thawed by letting them
stand at room temperature (15–25°C) and then centrifuged to separate the hemolyzed blood
cells and plasma components. Plasma was collected from thawed tubes.
DNA was extracted from 1.5 ml plasma using a Custom NEXTprep cfDNA Auto
Kit (1.5 ml; PerkinElmer, Waltham, MA, USA) with a Chemagic 360
instrument (PerkinElmer). The extract was dissolved in Milli-Q water (Merck Millipore,
Burlington, MA, USA) to a final volume of 50 µl.
4
SARS-CoV-2 Detection by RT-PCR
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All patient samples were tested for SARS-CoV-2 using an assay based on RNA extraction followed by TaqMan-based RT-PCR assay to conduct in vitro transcription of SARS-CoV-2 RNA, DNA amplification, and fluorescence detection (PerkinElmer Inc., Waltham, MA, USA). The assay targets specific genomic regions of SARS-CoV-2, the nucleocapsid (N) gene and ORF1ab with an RNA internal control (IC, bacteriophage MS2), to monitor the processes from nucleic acid extraction to fluorescence detection. The probes are labeled with FAM, ROX, and VIC dyes to differentiate the fluorescent signals from each target. The assay validation was performed as per FDA guidelines, following the manufacturer’s protocol. In brief, a 300 µL sample was used for RNA extraction (chemagic 360 instrument, PerkinElmer Inc., Waltham, MA, USA), to which 5 µL internal control, 4 µL poly(A) RNA, 10 µL proteinase K, and 300 µL lysis buffer 1 were added. From 60 µL eluate, the RT-PCR reaction was set up, which included 10 µL of extracted nucleic acid and 5 µL of PCR master mix. PCR was performed using QuantStudio 3 and 5 Real-Time PCR Systems (Thermo Fisher Scientific, Waltham, MA, USA). The LoD of this assay is 20 copies/mL.
5
CETP Gene Variants and HDL-C Levels
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Three SNPs—rs3764261 (-2568A/C), rs4783961 (-998A/C), and rs1800775 (-629A/C)—located in the promoter region (positions: chr16: 56959412, 56960982, 56961324 bp, respectively), as well as a missense coding SNP rs5882 (+16A/G) in exon 14 (position: chr16, 56982180 bp) of the CETP gene were included. The SNPs were selected from the human genome database (accessed at https://www.ncbi.nlm.nih.gov/genome/guide/human/ ) and, based on previously published reports, have been associated with an effect on HDL-C levels [9 (link),10 (link),22 (link),23 (link)]. SNPs were excluded if their minor allele frequencies in the HapMap CHB population were <0.01, or call rates were <98%. Additionally, SNPs that deviated from the Hardy–Weinberg equilibrium (HWE) were excluded (p < 0.05 indicated deviance from HWE). T2D subjects were genotyped using the Hap550K-BeadChip (Illumina, San Diego, CA, USA), which has been used previously for genome-wide association studies in the Han Chinese population of Taiwan [24 (link)]. For the non-diabetic controls from TWB, DNA was isolated from blood samples using a Chemagic™ 360 instrument following the manufacturer’s instructions (PerkinElmer, Waltham, MA, USA). SNP genotyping used custom-designed 653K TWB chips and was conducted on the Axiom Genome-Wide Array Plate System (Affymetrix, Santa Clara, CA, USA) [21 (link)].
6
Plasma-based MFQPCR Detection of Equine Muscle Genes
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Blood was collected from 264 Thoroughbreds into BD Vacutainer spray-coated
K2EDTA tubes (BD Biosciences, Franklin Lakes, NJ, U.S.A.). Plasma was separated
by centrifugation at 1,500 × g for 10 min. The separated plasma samples
were stored at −30°C. The separated plasma samples from the 264 horses were used for
MFQPCR detection. Among these samples, two plasma samples (1.5 ml) were
spiked with either 150 or 1,500 copies of Control_A_MSTN as positive detection models
(Table 1 Preparation of spiked samples for microfluidic quantitative PCR<br/>detection
Control_C_SET1 as contamination models of PTCs (Table
1 ). DNA was extracted from the 1.5 ml plasma samples using a
Custom NEXTprep cfDNA Auto Kit (1.5 ml; PerkinElmer, Waltham, MA, U.S.A.)
with a chemagic 360 instrument (PerkinElmer). The extract was dissolved in Milli-Q water
to a final volume of 50 µl.
K2EDTA tubes (BD Biosciences, Franklin Lakes, NJ, U.S.A.). Plasma was separated
by centrifugation at 1,500 × g for 10 min. The separated plasma samples
were stored at −30°C. The separated plasma samples from the 264 horses were used for
MFQPCR detection. Among these samples, two plasma samples (1.5 ml) were
spiked with either 150 or 1,500 copies of Control_A_MSTN as positive detection models
(
| Spiked sample | Content of plasma (ml) | Content of spiked control |
|---|---|---|
| Sample 1 | 1.5 | 150 copies of Control_A_MSTN |
| Sample 2 | 1.5 | 1,500 copies of Control_A_MSTN |
| Sample 3 | 1.5 | 150 copies of Control_C_SET1 |
| Sample 4 | 1.5 | 1,500 copies of Control_C_SET1 |
| Samples 5–264 | 1.5 | None |
Samples 1 and 2: positive detection models of single transgene. Samples 3 and 4:
contamination models of positive template controls.
Control_C_SET1 as contamination models of PTCs (
1
Custom NEXTprep cfDNA Auto Kit (1.5 ml; PerkinElmer, Waltham, MA, U.S.A.)
with a chemagic 360 instrument (PerkinElmer). The extract was dissolved in Milli-Q water
to a final volume of 50 µl.
7
COVID-19 and Influenza A(H1N1) Diagnosis
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A nasopharyngeal swab was collected from the patient, and nucleic acid extraction for the clinical sample was performed using the chemagic 360 instrument (PerkinElmer Inc). SARS-CoV-2 RNA (ORF1ab) was detected using a VIASURE SARS-CoV-2 Real-Time PCR Detection Kit (Certest Biotec SL). The RT-PCR runs were performed in triplicate and according to the manufacturer’s recommendations, and the samples were confirmed to be positive for SARS-CoV-2 using a cobas 6800 system (Roche Holding AG). Moreover, influenza A(H1N1) was tested by real-time PCR using the US Centers for Disease Control protocol [4 ]. A complete blood count (CBC) and computed tomography (CT) images of the chest were obtained for the patient.
8
SARS-CoV-2 Detection via Automated Nucleic Acid Extraction
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In routine screening, a batch of 94 samples were processed for the detection of SARS-CoV-2. In brief, an aliquot of 300 μL from each sample, including positive and negative controls, was added to respective wells in a 96-well plate. To each well, 5 μL internal control, 4 μL poly(A) RNA, 10 μL proteinase K, and 300 μL lysis buffer 1 were added. The plate was placed on a semiautomated instrument (chemagic 360 instrument; PerkinElmer, Inc.) following manufacturer's protocol. The nucleic acid was extracted in a 96-well plate, with an elution volume of 60 μL. From the extraction plate, 40 μL of extracted nucleic acid and 20 μL of RT-PCR master mix were added to the respective wells in a 96-well PCR plate. The PCR method was set up as per manufacturer's protocol on Quantstudio3 (Thermo Fisher Scientific, Waltham, MA). The samples resulted as positive or negative based on the Ct values specified by the manufacturer (Supplemental Table S1 ).
9
Saliva-based SARS-CoV-2 Detection Protocol
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All 879 saliva samples were tested using the FDA-EUA approved assay. In brief, the saliva samples were collected in 2 mL Omni tubes (2 mL reinforced tubes, SKU: 19-628D, Omni International, USA) and homogenized at 4.5 m/s for 30 s using the Omni bead mill homogenizer (Bead Ruptor Elite, SKU: 19-040E, Omni International, USA). An aliquot of 300 μL from each sample, positive and negative controls, was then added to respective wells in a 96 well plate. A 5 μL internal control (IC), 4 μL Poly(A) RNA, 10 μL proteinase K and 300 μL lysis buffer were then added to each well. The plate was placed on a semi-automated instrument (Chemagic 360 Instrument, PerkinElmer Inc.) following the manufacturer’s protocol. The nucleic acid was extracted in a 96 well plate, with an elution volume of 60 μL. From the extraction plate, 10 μL of extracted nucleic acid and 5μL of PCR master mix (3.75 μL reagent A, 0.75 μL reagent B, and 0.5 μL enzyme) were added to the respective wells in a 96 well PCR plate. The PCR method was set up as per the manufacturer’s protocol on Quantstudio 3 or 5 (ThermoFisher Scientific, Waltham, MA, USA). The samples were resulted as positive or negative, based on the Ct values specified by the manufacturer (Supplementary file 1 ).
10
SARS-CoV-2 RT-PCR Diagnostic Protocols
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Clinical swab and saliva specimens were previously collected under Johns Hopkins IRB #00246027. Specimens were de-identified and blinded before testing. Nasopharyngeal swabs were eluted in 3 mL of Universal Transport Medium. Passive drooled saliva specimens were collected into an empty vessel. Five microliters of saliva was lysed with 50 μL of aqueous buffer containing 1% Triton X-100 and 1.2 units of Thermolabile Proteinase K (P8111S, New England Biolabs). 50 μL of swab eluate or 55 μL of saliva with lysis buffer was mixed with 150 μL magnetic bead binding buffer as previously described followed by loading the entire mixture into the sample port of the cartridge. The comparator assay for evaluation of clinical samples used a modified CDC testing protocol32 for swabs (Supplementary Methods , Supplementary Fig. 8 ) and FDA EUA authorized SalivaDirect protocol43 for saliva were employed to test all the clinical specimens on a Bio-Rad CFX96 Touch Real-Time PCR System as reference.
Testing of SARS-CoV-2 variants used RNA from clinical samples extracted using a chemagic 360 instrument (PerkinElmer) with clades of each sample previously characterized by sequencing31 . 2 μL of extracted RNA was mixed with 150 μL magnetic bead binding buffer following by loading into the sample port of the cartridge.
Testing of SARS-CoV-2 variants used RNA from clinical samples extracted using a chemagic 360 instrument (PerkinElmer) with clades of each sample previously characterized by sequencing31 . 2 μL of extracted RNA was mixed with 150 μL magnetic bead binding buffer following by loading into the sample port of the cartridge.
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