Blood rna extraction kit

Total RNA was extracted from the proband, her parents, and a healthy control using a blood RNA extraction kit (QIAGEN). The extracted RNA was reverse transcribed into cDNA. qRT-PCR was then performed. The primer pair sequences were as follows: forward (5′-CAC​GTC​CAT​TGG​CTT​TCA​TC-3′) and reverse (5′-CCT​CAA​TCT​GCT​TAT​CCT​GGA​AGA-3′). GAPDH was used as a reference.

Whole blood RNA was extracted manually from PAXGene tubes, following manufacturer’s instructions (Blood RNA extraction kit, Qiagen, France). After extraction, samples were inactivated at 65°C for 5min then stored at -80°C until RT-qPCR. RT-qPCR was done using the qScript XLT One-Step RT-qPCR mix following manufacturer’s instructions (Quanta BioSciences, Inc., United States). Taqman probes (Applied Biosystems, ThermoFisher Scientific, France) and primers (Eurofins, France) for IFN-α1, IFNα2 and IFNα3 were described previously for P. alecto bat species in (30 (link)) and used here. Probes and primers for the bat GAPDH housekeeping gene were designed using Primer-BLAST from NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are presented in Table S2. All data were normalized relative to the housekeeping gene (GAPDH) as indicated. The expression level of the target genes was calculated using the standard curve method and expressed as copy numbers relative to the housekeeping gene.