To quantify CDV loads and cytokine responses at the molecular level, RT-qPCR analysis (38 slices derived from 4 dogs) was performed. RNA was isolated and purified from frozen PCLSs using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) as described [38 (link)]. Measurement of the optical density at 260 nm with a spectrophotometer (Multiskan™ GO microplate spectrophotometer, µDrop™ plate, SkanIt™ software version 5.0.0.42, Thermo Fisher Scientific, Braunschweig, Germany) allowed calculation of the obtained RNA amount and absorbance at 260 nm and 280 nm was used to assess the purity of RNA.
Total RNA was transcribed into complementary DNA (cDNA) using the Omniscript® Reverse Transcription Kit (Qiagen), RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, Carlsbad, CA, USA), and random primers (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions as described [38 (link)].
Primer sequences and plasmids for generation of standard dilutions and qPCR products (Eurofins Genomics, Ebersberg, Germany) were taken from the literature and have been described in detail in a previous publication [38 (link)]. Standard dilutions were either generated from extracted PCR products or plasmids, and RT-qPCR analysis using the AriaMx Real-Time PCR System (Agilent Technologies; Agilent Aria software version 1.71) was performed as described [38 (link)].
Total RNA was transcribed into complementary DNA (cDNA) using the Omniscript® Reverse Transcription Kit (Qiagen), RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, Carlsbad, CA, USA), and random primers (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions as described [38 (link)].
Primer sequences and plasmids for generation of standard dilutions and qPCR products (Eurofins Genomics, Ebersberg, Germany) were taken from the literature and have been described in detail in a previous publication [38 (link)]. Standard dilutions were either generated from extracted PCR products or plasmids, and RT-qPCR analysis using the AriaMx Real-Time PCR System (Agilent Technologies; Agilent Aria software version 1.71) was performed as described [38 (link)].
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