title: Bio-Rad CFX96 Touch Real Time PCR Detection System [Ferret]
status: Public on Apr 01 2022
contact.city: Madison
contact.first: Bryan
contact.last: Zabel
contact.email: bryan.zabel@iff.com
contact.state: wi
contact.address: 3329 Agriculture Dr
contact.department: GEM
contact.country: USA
contact.institute: International Flavors & Fragrances Inc
organism: Mustela putorius furo
description: PCR assay
manufacture_protocol: The total RT reaction volume was 50 µL and RT was performed in a 96 well plate on a Bio-Rad CFX96 Touch Real Time PCR Detection System. After RT, the cDNA was diluted with nuclease free water to bring the final total volume up to 200 µL (4x dilution). 5 µL of diluted cDNA was used for qPCR analysis of the expression of gene of interest. The total PCR reaction volume was 15 µL per well (10 µL master mix + 5 µL cDNA sample) and the qPCR was performed on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using the cycling conditions listed below:
  Temperature
  95°C; 2 minutes; 1 Cycle(s)
  95°C; 10 seconds; 40 Cycle(s)
  *60°C or 62°C; 45 seconds
  *60 °C for CCL2, IFNα, IFNL1 and IL8
  62 °C for all other genes (CCL5, CXCL10, IFNγ, IL10, IL1β, IL6, TLR8, TNFα, ACE2 and RPS18)
  Each plate contains a positive control (prepared from stimulated ferret PBMC cells) in duplicate wells, an NTC in duplicate wells and all test samples in duplicate wells. Primer and probe sequences can be found in Supplemental Table 3.