We then extracted the RNA from 400 [micro]L of the filtered and unfiltered aliquots of seminal plasma and quantified the 5' region of the ACTB and DDX4 mRNAs.
Short PCR amplicons (<240 bp; see Table 1 in the online Data Supplement) targeting different regions of the ACTB and DDX4 transcripts were detected in cfsRNA samples from all 9 volunteers; however, we detected long PCR amplicons of ACTB mRNA (1499 bp) in 2 of the 9 volunteers but detected no long amplicons for DDX4 mRNA (1909 bp).
We further evaluated the integrity of cfsRNA by quantifying the amounts of the 3 different regions of ACTB and DDX4 transcripts.
Given that cfsRNA exists mainly in partially degraded forms and that human semen is known to contain various ribonucleases (25), we tested the stability of cfsRNA by a time-course analysis of different regions of the ACTB and DDX4 transcripts.
4, A and B, revealed no significant changes in seminal ACTB mRNA or DDX4 mRNA concentrations after filtration through a 5-[micro]m filter, suggesting that these mRNA transcripts were not contained within intact cells.
3%, respectively, of the 5' region of ACTB mRNA and could not amplify any DDX4 mRNA (Fig.
Detection of full-length transcripts was rare, and the quantitative analysis of multiple regions of seminal mRNAs showed preferential degradation of the 3' region for both ACTB and DDX4, indicating that exonucleases were primarily involved in the degradation of cfsRNA.
The 3' region of DDX4, however, was scarcely detectable and underwent more rapid degradation.