The pWR3.26 molecular clone was obtained from the ATCC to rescue HRV-14 viral stocks, while the pR16.11 plasmid was obtained from the ATCC to generate HRV-16 viral stocks (GenBank accession no. L24917 ). Supernatants containing infectious virus for both HRV-16 and HRV-14 were isolated and aliquoted. The GenBank reference sequence for HRV-16 was used to design a quantitative reverse transcriptase PCR (qRT-PCR) TaqMan primer/probe set: TaqMan forward primer, 5′-GGTTAAATGGATGTTAAGAATTATATCAGCTATGGTTATA-3′; TaqMan reverse primer, 5′-CATCCAATCAGTGTTAAAGTGGCAAT-3′; TaqMan probe, 6-carboxyfluorescein (FAM)-CAGATCCGCAAACAAT. To isolate RNA, supernatant after each passage was frozen at −80°C and subsequently subjected to RNA extraction using a Qiagen RNeasy kit. RNA was treated with RNase-free DNase I. Reverse transcription was performed with Superscript III reverse transcriptase (Invitrogen). As a negative control to monitor contamination, water was used in place of Superscript III reverse transcriptase, and in each case, reverse transcription was performed with oligo(dT) priming. cDNA was diluted to 1:100 and run in duplicate using the TaqMan custom HRV-16 custom primers and probe on the Applied Biosystems 7500 platform.
7500 platform
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7500 platform is a real-time PCR system designed for a variety of applications, including gene expression analysis, SNP genotyping, and pathogen detection. The system features a 96-well format, multiple color detection, and a compact footprint. The 7500 platform provides a reliable and efficient solution for DNA and RNA analysis in research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Ultrapure bsa, by Thermo Fisher Scientific (1 mentions)
Power sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Chemagic msm 1, by PerkinElmer (1 mentions)
Taqman genotyping assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using 7500 platform
1
Quantification of Human Rhinovirus Strains
2
Quantifying Bacterial 16S rRNA Genes
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Copy numbers of 16S ribosomal genes were determined by qPCR using the Applied Biosystems 7500 platform. Each 20 μL qPCR reaction contained 10 μL of Power SYBR Green Master Mix (Applied Biosystems, Life Technologies), 0.2 μL of the forward Eub338 (5’-ACTCCTACGGGAGGCAGCAG-3’) and reverse Eub518 (5’-ATTACCGCGGCTGCTGG-3’) primers at a concentration of 10 μM, 2 μL bovine serum albumin (Ambion Ultrapure BSA, 5 mg mL–1), 2 μL of template DNA and 5.6 μL sterile water [42 (link)]. All PCR reactions were performed in triplicate using 8 ng template DNA for each experimental unit and were performed as described in Gleeson et al. [43 ]. Standard curves generated in each reaction were linear over four orders of magnitude (104 to 107 gene copies) with R2 values greater than 0.99. Efficiencies for all quantification reactions were 90–100%.
3
Analyzing EbFS Gene Expression in Transgenic Chrysanthemum
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For the analysis of EbFS gene expression in transgenic chrysanthemum plants, total RNA was isolated using Trizol from leaf, flower stem, and flower head tissues at different developmental stages, as well as after mechanical damage treatment. For the mechanical damage treatment, five flowers were selected and subjected to rapid puncturing ten times using entomological pins on the flower stem. Subsequently, the wounded and unwounded flower stems (3 centimeters below the flower head) were cut and immediately flash-frozen in liquid nitrogen. The RNA samples were reverse transcribed as described previously (Li et al., 2019 (link)). Real-time gene expression analysis of EbFS and the reference gene CmUBI was performed on an Applied Biosystems 7500 platform using SYBR Green I with 6-carboxyl-X-rhodamine (ROX) as an internal standard, following the protocol described by Li et al. (Li et al., 2019 (link)).
4
Quantifying Viral Loads in Nasal Secretions
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DNA was isolated from nasal swab samples to determine viral genome copy numbers in nasal secretions by quantitative PCR targeting the gB gene [37 ] using purified amplicon standards and 4 μl of template (from a 90 μl elution). Aliquots of 5 x 106 snap frozen PBMC pellets were extracted and tested using the same assay with a positive cutoff imposed at the analytic limit of 100% detection. Cycle threshold (Ct) values were calculated based on the automated algorithm on the Applied Biosystems 7500 platform. The quantitative PCR was performed at the Animal Health Diagnostic Center at Cornell University.
5
SARS-CoV-2 Detection in Nasopharyngeal Samples
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Nasopharyngeal specimens were obtained by trained university health staff observing standard procedures and stored in 2 mL of viral transport media (VTM) containing 1X Hanks Balanced Salt Solution, 2% Fetal Bovine Serum, 0.1 mg/mL gentamicin, and 0.5 µg/mL amphotericin B. All samples were transported on ice in secondary containers to the testing laboratory and processed within a four-hour timeframe. A modified version of the CDC Emergency Use Authorization (EUA) real-time RT-PCR protocol [8 ] was used to detect SARS-CoV-2 in specimens using the Applied Biosystems 7500 platform (Waltham, MA, USA). Specimens were resuspended in VTM by vortexing for 10 sec and then transferring 500 µL to an Eppendorf tube. Boiling samples at 368.15 K for 5 min was utilized to extract RNA from specimens [9 (link)].
6
Cognitive Assessment and Genetic Profiling in FINGER
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The present study used data from the FINGER baseline visit, before the start of the intervention. Cognition was measured using a modified version of the Neuropsychological Test Battery (mNTB) (Harrison et al., 2007 (link)). A standardized composite mNTB score was determined based on 14 individual tests measuring three different cognitive domains, i.e., memory, executive function, and processing speed. Domain-specific standardized mNTB scores were also calculated as previously described (Ngandu et al., 2015 (link)), with higher scores indicating better performance. Height, weight, and blood pressure were measured (Ngandu et al., 2014 (link)), and body mass index (BMI) and hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg) were used as vascular risk factors in the model. Genomic DNA was extracted from venous blood samples with Chemagic MSM1 (PerkinElmer) using magnetic beads. APOE genotype was determined by polymerase chain reaction using TaqMan genotyping assays (Applied Biosystems) for two single-nucleotide polymorphisms (rs429358 and rs7412) and an allelic discrimination method on the Applied Biosystems 7500 platform (De La Vega et al., 2005 (link)).
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