Absolute Gene Expression Analysis of fads2 in PBCs and Liver

Total RNA extraction from peripheral blood cells and liver were made using a similar protocol described above for the qPCR. Absolute gene expression of PBCs and liver fads2 analysis were performed using Digital Droplet PCR (ddPCR) (Bio-rad QX200, Hercules, CA, USA) systems, by using cDNA obtained as mentioned in Section 2.3. Sample preparation for ddPCR was carried out as per the manufacturer’s protocol. The master mixes for fads2 gene were prepared including 10 μL EvaGreen super mix (Bio-rad, Hercules, CA, USA), 0.2 μL F primer (10 pmol/μL), 0.2 μL R primer (10 pmol/μL), 7.6 μL MilQ water, and 2 μL cDNA. Then, droplets were generated using droplet generator Bio-rad QX200 (Hercules, CA, USA) and the droplets were transferred to 96 well microplates for PCR in a thermal cycler (Bio-rad C1000 Touch, Hercules, CA, USA). After PCR amplifications, droplets were measured with a droplet reader (Bio-rad QX200, Hercules, CA, USA) to determine absolute gene expression of fads2 gene. The samples with less than 12,000 droplets were not used for the gene expression study. The fads2 gene expression analysis was performed in two replicates for each sample and values were expressed as mRNA copies/µL [31 (link),33 (link)].