RNA was isolated from frozen PBMCs using the RNeasy Plus kit; to
maximize genomic DNA elimination, the samples went through the gDNA eliminator
with on-column DNase digestion (Qiagen, Venlo, Netherlands). First-strand cDNA
synthesis was performed with random hexamers using SuperScript III First-Strand
Synthesis System (Thermo Fisher, Waltham, MA). Polymerase chain reaction was
performed using heat-activated immomix (Bioline, London, UK). To prevent carry
over DNA contamination, dUTP was spiked into each PCR reaction, and all
subsequent PCR reactions underwent an initial uracil-DNA glycosylase incubation
step (Amresco, Solon, OH). Gene-specific primers were created using sequences
from Canis Familiaris GenBank reference sequence DQ156494.1 for SMCY and
sequence AF107021.1 for SRY. All the primer sets spanned introns with the
exception of the SRY gene, which has no introns.
maximize genomic DNA elimination, the samples went through the gDNA eliminator
with on-column DNase digestion (Qiagen, Venlo, Netherlands). First-strand cDNA
synthesis was performed with random hexamers using SuperScript III First-Strand
Synthesis System (Thermo Fisher, Waltham, MA). Polymerase chain reaction was
performed using heat-activated immomix (Bioline, London, UK). To prevent carry
over DNA contamination, dUTP was spiked into each PCR reaction, and all
subsequent PCR reactions underwent an initial uracil-DNA glycosylase incubation
step (Amresco, Solon, OH). Gene-specific primers were created using sequences
from Canis Familiaris GenBank reference sequence DQ156494.1 for SMCY and
sequence AF107021.1 for SRY. All the primer sets spanned introns with the
exception of the SRY gene, which has no introns.