Selected article for: "master mix and synthesis kit"
Author: Van der Gucht, Winke; Stobbelaar, Kim; Govaerts, Matthias; Mangodt, Thomas; Barbezange, Cyril; Leemans, Annelies; De Winter, Benedicte; Van Gucht, Steven; Caljon, Guy; Maes, Louis; De Dooy, Jozef; Jorens, Philippe; Smet, Annemieke; Cos, Paul; Verhulst, Stijn; Delputte, Peter L.
Title: Isolation and Characterization of Clinical RSV Isolates in Belgium during the Winters of 2016–2018
  • Document date: 2019_11_6
    • ID: 0imlae98_23
    Snippet: A549 cells were seeded at a concentration of 200,000 cells/well in 24-well plates, 24 h prior to inoculation (Greiner bio-one). Cells were infected with a MOI of 0.1 for 2 h at 37 • C (5% CO 2 ). After 2 h, inoculum was replaced by DMEM-10 and was incubated for an additional 48 h. Afterwards, cell supernatant was collected, spun down at 1000× g for 15 min and only the pellet was kept. The still adherent cells were lysed with lysis buffer from .....
    Document: A549 cells were seeded at a concentration of 200,000 cells/well in 24-well plates, 24 h prior to inoculation (Greiner bio-one). Cells were infected with a MOI of 0.1 for 2 h at 37 • C (5% CO 2 ). After 2 h, inoculum was replaced by DMEM-10 and was incubated for an additional 48 h. Afterwards, cell supernatant was collected, spun down at 1000× g for 15 min and only the pellet was kept. The still adherent cells were lysed with lysis buffer from the nucleospin kit (Macherey-Nagel; Düren, Germany) and added to the pellet. The solution was thoroughly mixed and frozen at -80 • C until extraction was performed. RNA isolation was done following manufacturer's instructions of the nucleospin RNA kit (Macherey-Nagel). Concentrations were evaluated using the Nanodrop ®® (Thermo Fisher Scientific) and 1 µg of RNA was used to convert to cDNA using the SensiFast™ cDNA synthesis kit (Bioline; London, UK). Relative gene expression was determined with the GoTaq qPCR master mix (Promega; Madison, WI, USA) with SYBR Green Fluorescence detection on a QuantStudio 3 Real-time PCR instrument (Thermo Fisher Scientific). Standard QuantiTect primers available from Qiagen were used for GAPDH (QT00079247), ß-actin (QT00095431), MUC1 (QT00015379), MUC4 (QT00045479), MUC5AC (QT00088991) and MUC5B (QT01322818). Analysis and quality control were performed using qbase+ software (Biogazelle; Ghent, Belgium), relative expression of the target genes was normalized to the expression of the housekeeping genes GAPDH and ß-actin.

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