Clinical specimens of nasopharyngeal swabs were collected from patients in New York City during the 2014–2015 flu season as previously described [24 (link)]. The specimen used in this study was designated as A/New York/A39/2015 (H3N2) and is available in the SRA as sample ID SAMN08454624. Briefly, the RNA was eluted in 30 µL of RNase-free water and 3 µL was used as a template for the amplification of the entire influenza A or B genome using a previously described multi-segment RT-PCR method [35 (link)]. The presence of the cDNA copies of the genomic segments was examined by running 3 µL of the multi-segment RT-PCR amplicons on a 0.8% agarose electrophoresis gel. The influenza genomic amplicons were purified using a 1x Agencourt AMPure XP purification step (Beckman Coulter, Indianapolis, IN), and assessed by Qubit analysis (ThermoFisher, Waltham, MA) to quantify the mass of the double-stranded cDNA present.
Qubit analysis
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit analysis is a laboratory instrument used for quantifying and analyzing DNA, RNA, and protein samples. It provides accurate and sensitive measurements of sample concentrations using fluorescent dyes that bind specifically to the target molecules. The Qubit analysis delivers precise results with a small sample volume, making it a valuable tool for researchers and scientists in various fields of study.
Automatically generated - may contain errors
3 protocols using qubit analysis
1
Influenza A Genome Amplification Protocol
2
In Vitro Transcription of Target RNAs
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Target RNAs were generated using MEGAshortscript T7 kit (ThermoFisher) with PCR templates. Products were gel-purified and quantified by Qubit analysis (ThermoFisher). IVT templates for Figures 4 and 6 C target RNAs were made by PCR of the annealed oligos 2397/2398 with primers 3110/3112 (t) or 3115/3114 (rc). For Figure 6 E–G IVT templates, target plasmids were amplified with primers 2798/2801. Plasmids pJE65 (GGG), pJE66 (rc-GGG), pJE67 (AAA), pJE69 (CCC), pJE71 (UUU), pJE294 (mut-GGG), pJE201 (UGG), pJE189 (UUG), pJE198 (UGU), pJE202 (CUU), pJE190 (UCU), and pJE187 (UUC) were used as PCR templates.
3
RNA Quality Assessment for Library Preparation
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Total RNA samples were subjected to three quality control analyses prior to library construction: (A) NanoDrop analysis (Thermo Fisher, United States) to assess RNA purity (OD260/OD280; OD260/OD230); (B) Qubit analysis (Thermo Fisher, United States) to determine the RNA yield; and (C) Agilent 2100 Bio-analyzer analysis (Agilent Technologies) to verify RNA integrity. The significance of the results was determined using analysis of variance (ANOVA) and SPSS Statistics software, version 24.0 (IBM, United States).
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