A contaminant-free assessment of Endogenous Retroviral RNA in human plasma

Abstract

Endogenous retroviruses (ERVs) comprise 6-8% of the human genome. HERVs are silenced in most normal tissues, up-regulated in stem cells and in placenta but also in cancer and HIV-1 infection. Crucially, there are conflicting reports on detecting HERV RNA in non-cellular clinical samples such as plasma that suggest the study of HERV RNA can be daunting. Indeed, we find that the use of real-time PCR in a quality assured clinical laboratory setting can be sensitive to low-level proviral contamination. We developed a mathematical model for low-level contamination that allowed us to design a laboratory protocol and standard operating procedures for robust measurement of HERV RNA. We focus on one family, HERV-K HML-2 (HK2) that has been most recently active even though they invaded our ancestral genomes almost 30 millions ago. We extensively validated our experimental design on a model cell culture system showing high sensitivity and specificity, totally eliminating the proviral contamination. We then tested 236 plasma samples from patients infected with HIV-1, HCV or HBV and found them to be negative. The study of HERV RNA for human translational studies should be performed with extensively validated protocols and standard operating procedures to control the widespread low-level human DNA contamination.

Figure 1. Multiple melt curves of HK2 pol molecular beacon probe against all possible proviral sequences.

Beacon alone in black, correct target (K113) in red, detectable targets in green, undetectable targets in blue.

Figure 2

Probability of getting at least one false positive reaction due to low-level contaminants (A) as a function of the probability of detecting the contaminant in any reaction and (B) as a function of the number of negative controls included in an experiment with 32 reactions.

Figure 3. An example of an amplification curve for molecular beacon qPCR for HK2 pol.

Purple: 10⁴ RNA standard, light blue: 10² RNA standard, green: RT+/RT− samples (N = 14), pink: negative control.

Figure 4. RNA Standard Curve.

Log10 serial dilutions of DNase-treated 10⁸ RNA standard, tested in duplicate. Threshold cycle (Ct) values are plotted against the number of RNA copies/reaction.