7300 rt pcr machine

Viral RNA load was assessed in tissues from the dam, fetus, and placenta using a ZIKV-specific RT-qPCR assay. Fetal and maternal organs were immersed in RNA-later immediately upon harvest and were then weighed and homogenized in RLT solution (Qiagen) using a bead-beater apparatus (Precellys). RNA was extracted from tissues using the RNeasy kit (Qiagen) and from sera using the ZR Viral RNA extraction kit (Zymo Research). From tissues, 400 ng of total RNA was used to generate cDNA using the iScript select cDNA synthesis kit (Bio-Rad) according to manufacturer’s protocols for gene-specific primers. Viral RNA was quantified using the Taqman Universal Master Mix (Applied Biosystems) and an Applied Biosystems 7300 RT-PCR machine with primers that correspond to residues conserved in both the FSS13025 and Brazil Fortaleza genome (GenBank numbers KU955593.10, KX811222.1).^{49 (link)} To adhere to stringent guidelines, Ct (cycle threshold) values >38 were deemed as not reliably detected and were not reported. Copy number sensitivity, as determined using a standard curve from diluted known quantities of ZIKV genome, was 25 copies/qPCR reaction.

Thymi from wild-type mice were dissociated using collagenase (Sigma-Aldrich.) digestion for 1 h at 37°C. Some sample was reserved for RNA isolation (“whole thymocyte” samples), and the remainder was used for DC enrichment using MACS CD11c microbeads according to manufacturer’s instructions (Miltenyi), followed by flow cytometry to reach a final purity >85% CD11c⁺ F4/80⁻ population (“thymic DC” samples). The thymus sample after removal of CD11c⁺ cells was also used for RNA isolation (“CD11c depleted” samples). Cell samples were lysed in Trizol (Life Technologies), and total RNA prepared using the RNAeasy Kit (Qiagen) as per manufacturer’s instructions. Reverse transcription was performed using the Quatitect RT Kit (Qiagen) and cDNA was utilized to run qPCR reactions using Taqman probes along with TaqMan Real-Time PCR master mix (Life Technologies). PrimeTime probes, (Mm.PT.58.11478202 (IL-2) and Mm.PT.39a.1 (GAPDH)), were synthesized by IDT. All reactions were run on an Applied Biosystems 7300 RT PCR machine. IL-2 expression data were normalized to GAPDH and expressed as fold increase over the background values from Il2^−/− bone marrow-derived DCs using ΔΔC_t values. (For Il2^−/− DCs, no IL2 signal was observed after 40 cycles of PCR, therefore values reported are upper estimates (indicated by grey shading), and were used to determine the lower limit of detection of the assay.