China
Africa
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Bacognize
Published in Dilip Ghosh, Pulok K. Mukherjee, Natural Medicines, 2019
Molecular diagnostic test: The DNA concentration is assessed using a Qubit Fluorometer in addition to the TRU-ID DNA authentication test using TRU-ID mini-sequence analysis technology. Molecular diagnostics in this test uses Sanger sequencing of standardised DNA sequences of a plant. Samples are analysed against TRU-ID Plant DNA library.
Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression
Published in OncoImmunology, 2023
Paul G. Pavicic, Patricia A. Rayman, Shadi Swaidani, Amit Rupani, Vladimir Makarov, Charles S. Tannenbaum, Robert P. Edwards, Anda M. Vlad, C. Marcela Diaz-Montero, Haider Mahdi
Cells isolated from peritoneal lavages of i.p. ID8-VEGF tumor-bearing mice that were in relapse or remission following intervention with dual-ICI + IL12 were subjected to single-cell RNA sequencing. Single-cell suspensions were prepared, loaded on the 10× Genomics Chromium System (Single Cell 3’ v3 platform) and libraries prepared according to manufacturer’s specifications. The concentration and quality of double-stranded cDNA was assessed using a high-sensitivity DNA assay on an Agilent 2100 Bioanalyzer. Library preparation for sequencing on an Illumina platform was accomplished for each sample following the manufacturer’s protocol (10× Genomics, CG000183 Rev A). The quality of library construction was again assessed using the Agilent 2100 Bioanalyzer. Samples were first fluorometrically quantified with a Qubit fluorometer (Thermo Fisher Scientific), pooled, and again quantified on a Quantabio Q cycler using the Quantabio SparQ Fast Library Quant kit. Sequence data was generated on a NovaSeq 6000 using parameters recommended by the manufacturer (10× Genomics, CG000183 Rev A). The bioinformatics of output data was performed using Cell Ranger software (10× Genomics) mkfastq, count, and AGGR functions. Subsequently, Cell Loupe browser (10× Genomics) files were generated, and data visualized by UMAP, individual gene, and heat map plots.
Comparison of Intraocular Antibody Measurement, Quantitative Pathogen PCR, and Metagenomic Deep Sequencing of Aqueous Humor in Secondary Glaucoma Associated with Anterior Segment Uveitis
Published in Ocular Immunology and Inflammation, 2022
Li Wang, Zhujian Wang, Jinmin Ma, Qiongfang Li, Xueli Chen, Yuhong Chen, Xinghuai Sun
Total RNA nucleic acids were extracted from 50 μl aqueous humor by phenol-chloroform method with TRIZOL. Briefly, nucleic acids (RNA) were reversely transcribed to complementary DNA (cDNA) using random primers and then amplified using a multiple displacement amplification (MDA) random-amplification approach (REPLI-g Single Cell WTA kit, Qiagen, Germany).21,22 Genomic library construction and next-generation sequencing (NGS) were conducted according to the BGI-Seq500 sequencing protocol. Amplified cDNA was purified using AmPure beads (Qiagen, Germany). The products were then quantitated by Qubit Fluorometer 3.0 (Life Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies Inc. USA). A total of about 1 μg of amplified products was fragmented into 250 bps by Covaris E210 (Covaris, USA) and then was constructed into the library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle amplification. The DNBs were loaded on the patterned nanoarrays and sequenced on BGI-Seq500 platform (BGI, China) using a single-end 100 bp sequencing.
Development of the Next Generation Sequencing-Based Diagnostic Test for β-Thalassemia and its Validation in a Pashtun Family
Published in Hemoglobin, 2020
Bibi Sabiha, Syed Adnan Haider, Hanifullah Jan, Yasar Mehmood Yousafzai, Ome Kalsoom Afridi, Abid Ali Khan, Johar Ali
The HBB gene on chromosome 11, was amplified using a set of primers encompassing a 1900 bp stretch of DNA starting from 5,225,298-5,227,197 coordinates on the reverse strand. The primer set for HBB was designed using Genome Reference Consortium (GRC), GRCh38 (also called ‘build 38′) with the help of Primer3 software available at http://bio info.ut.ee/primer3-0.4.0/. The primers used for the amplification of the HBB are given in Table 1. The PCR was carried out using the Thermo Scientific™ Phusion™ Flash High-Fidelity PCR Master Mix (Cat #F-548S; Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A 2.5 µL of each amplified product was run on a 1.5% agarose gel to check the product amplification. The PCR product of each sample was quantified by Qubit fluorometer using dsDNA High Sensitivity kit (Qubit, Cat. #Q32851; Invitrogen) and normalized to a concentration of 0.2 ng/µL.