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. 2020 Feb 28;94(6):e01441-19. doi: 10.1128/JVI.01441-19

FIG 12.

FIG 12

miRNA-specific qRT-PCR detection and quantitation of EBOV GP vncRNA in nonhuman primate liver tissue. Total RNA was extracted from archived liver tissues from EBOV-vaccinated and unvaccinated rhesus macaques and was subjected to miRNA-specific qRT-PCR. qRT-PCRs for unknowns were performed in triplicate; qRT-PCRs for serially diluted standards were performed in duplicate. Total RNA extracted from EpoNi/22.1 cells at 24 h after infection with wt rEBOV–eGFP was used as a positive control. A synthetic ssRNA oligonucleotide homologous to GP vncRNA was spiked into total human brain RNA at a concentration of 0.1 ng/μl (total human brain RNA concentration, 10 ng/μl) and was used as a standard for absolute quantitation (undiluted; 10−7). (A) Amplification curves for a wild-type human parainfluenza virus type 3 (HPIV3)-vaccinated control rhesus macaque (moribund animal euthanized 8 days postchallenge; extrapolated GP vncRNA copy number, 3.06 × 103 copies/ng total RNA), an HPIV3/EboGP-vaccinated rhesus macaque (surviving animal euthanized 28 days postchallenge, at study endpoint; extrapolated GP vncRNA copy number, 0 copies/ng total RNA), wt rEBOV–eGFP-infected EpoNi/22.1 cells at 24 hpi (extrapolated GP vncRNA copy number, 2.78 × 104 copies/ng total RNA), and the EBOV GP vncRNA standard (ssRNA GP Oligo) (10−3 dilution; 8.4 × 106 copies/ng total RNA). (B) Melt curve analysis of GP vncRNA qRT-PCR amplicons. For clarity, the melt curve for only a single technical replicate is shown for each sample and is representative of all technical replicates for that sample. The average amplicon Tm values of three technical replicates for each sample (two for the standard) were as follows: for the HPIV3-vaccinated control rhesus macaque, 70.45°C; for the HPIV3/EboGP-vaccinated rhesus macaque, 65.19°C; for EpoNi/22.1 total RNA at 24 h after infection with wt rEBOV–eGFP, 70.50°C; for the EBOV GP vncRNA standard (10−3 dilution; ∼8.4 × 105 copies/ng total RNA), 70.54°C. Rn, ratio of the fluorescence of the reporter dye (SYBR Green) to the fluorescence of the passive ROX dye. ΔRn, normalized Rn value obtained by subtracting the baseline fluorescence of the reporter dye from Rn. -R', negative derivative reporter, obtained as the first negative derivative of the the normalized Rn value. All values are relative fluorescent units reported and plotted by the Applied Biosystems StepOne software.