PSX-B-15 Real-time quantitative PCR versus droplet digital PCR for measurement of peripheral blood leukocytes interferon-tau stimulated genes in beef cattle

Abstract

Abstract We aimed to compare the abundance of interferon-tau stimulated genes (ISG) transcripts in peripheral blood leukocytes of artificially inseminated beef cows using real-time quantitative PCR (RT-qPCR) versus Droplet Digital PCR (ddPCR). Multiparous Bos taurus beef cows (n = 7) were subjected to timed artificial insemination (TAI) on day 0. Pregnancy was determined by transrectal ultrasonography on days 26 and 30 post-TAI, and cows were classified as: pregnant (n=4; embryo detected on days 26 and 30) or non-pregnant (n = 3; no embryo detected). Coccygeal vein blood samples were collected on days 0, 15, 17, 19, 20 and 24 post-TAI. Leukocyte RNA was extracted from the buffy coat fraction using Trizol associated with the DirectZol-RNA kit and transcribed to cDNA. The abundance of ISG (ISG15 and MX2) was assessed by relative quantification to a reference gene (RPS9) using RT-qPCR and by absolute quantification using the QX100TM Droplet DigitalTM PCR System (Bio-rad Laboratories) according to manufacturer’s recommendations. Data were analyzed using PROC MIXED on SAS 9.4. For the RT-qPCR, pregnant cows had greater (P < 0.05) ISG15 and MX2 abundance compared to non-pregnant cows on days 20 (ISG15:0.11±0.1 vs. 0.01±0.001 and MX2:0.73±0.4 vs. 0.06±0.06) and 24 (ISG15:0.34±0.2 vs. 0.01±0.001 and MX2:0.77±0.2 vs. 0.13±0.04). For ddPCR, a greater ISG15 and MX2 copy numbers in pregnant cows was observed on days 15 (ISG15:129 vs. 44 copies/µl and MX2:33 vs. 10 copies/µl) and 20 (ISG15: 216 vs. 30 copies/µl and MX2: 44 vs. 7 copies/µl), and also on day 24 for ISG15 (32 vs. 7 copies/µl) compared to non-pregnant cows. In conclusion, ddPCR was able to detect an earlier expression of ISG in pregnant cows. Future studies are needed to enroll more animals and establish a suitable cutoff value using ddPCR, which could be less subjective for diagnosis as it does not require the use of a reference gene.