Itaq universal probes one step kit

The mRNA levels of the target genes were quantified using real time-PCR (qRT-PCR) using TaqMan Universal PCR Master Mix, no AmpErase UNG (ThermoFisher Scientific, Waltham, MA). qRT-PCR was carried out at 48 °C for 30 minutes, 95 °C for 10 minutes, 95 °C for 15 seconds, and 60 °C for 1 minute (40 cycles of steps 3 and 4) on a CFX96 or CFX384 (Bio-Rad, Hercules, CA) sequence detection PCR system. The relative expression was calculated using the 2^−ΔΔCt analysis method with expression normalized to the housekeeping gene peptidylprolyl isomerase B (Ppib). All samples were analyzed in duplicate; qRT-PCR was carried out using 100 ng RNA. The expression of the following genes was evaluated: syndecan-1 (SDC1; Rn00564662_m1), syndecan-3 (SDC3; Rn00588067_m1), syndecan-4 (SDC4; Rn00561900_m1), glypican-1 (GPC1; Rn01290371_m1), and CD44 (CD44; Rn00681157_m1). All probes were purchased from Applied Biosystems (Waltham, MA). The abundance of syndecan-1 mRNA was determined using an iTaq Universal Probes One-Step Kit (Bio-Rad, Hercules, CA) and 200 ng RNA. The reaction was carried out at 50 °C for 10 minutes, 95 °C for 5 minutes, 95 °C for 10 seconds, and 60 °C for 30 seconds (40 cycles of steps 3 and 4).

Viral RNA was extracted from ejaculates and homogenized tissue samples using the QIAamp viral RNA mini kit (Qiagen). Briefly, 70μL sample was diluted 1:2 in 10μM dithiothreitol (DTT; Pierce) to denature seminal proteins. Samples were lysed in 560μL buffer AVL with linear acrylamide added (1μg per sample). The extraction was continued following the manufacturer’s protocol. Viral RNA was eluted in 60μL nuclease-free water (Qiagen) and stored at −80C.
Viral RNA was quantified via a one-step qRT-PCR using the iTaq universal probes one-step kit (Bio-Rad) per the manufacturer’s instructions for a 20μL reaction, with the exception that the quantity of reverse transcriptase per reaction was halved. Five microliters of RNA were used per reaction. ZIKV specific primers and probes were synthesized by IDT using 6-Fam as the reporter dye and Zen/Iowa Black as the quencher. Primer and probe sequences and cycling conditions are as previously described (41 (link)). The amplification product is an approximately 75bp region of the envelope protein. Viral RNA concentration was determined via an absolute standard curve of in vitro transcribed RNA standards from a plasmid containing a segment of the ZIKV envelope gene (20 (link), 41 (link)). The limit of detection for this assay was 2 log₁₀ RNA copies per ejaculate or 1.5 log₁₀ RNA copies per organ.