Viral rna dna mini kit

Total RNA was extracted from lungs of infected animals, using viral RNA/DNA Mini Kit (PureLink ® -Invitrogen), following the manufacturer's instructions. cDNA was synthesized with random primers using Sensiscript ® Reverse Transcription kit (QIAGEN ® ). The quality of cDNA for each sample and Quantitative real time PCR (qPCR) was performed as previously described (Freitas et al., 2016) . For the standard curve, ten-fold serial dilution of 6 × 10 7 copies of a plasmid with RSV F protein sequence were added to the same plate of qPCR in duplicate. The results were used for further quantification of viral load.

Total RNA was extracted from RSV infected BMDC using Viral RNA/DNA Mini Kit (PureLink® -Invitrogen) following the manufacturer's instructions. cDNA was synthetized with random primers using Sensiscript® Reverse Transcription kit (QIAGEN®). The quality of the cDNA for each sample was tested by amplification of β-actin endogenous gene using specific primers and probes from TaqMan Assay (Applied Biosystems®). Samples that did not amplify β-actin gene were excluded. Real time PCR was performed to RSV protein F gene amplification using specific primers and probes (forward 5′-AACAGATGTAAGCAGCTCCGTTATC-3′, reverse 5′-CGATTTTTATTGGATGCTGTACATTT-3′ and probe 5′-FAM/ TGCCATAGCATG ACACAATGGCTCCT-TAMRA/-3′). Data were analyzed by cycle threshold (CT). Delta CT value was calculated from the difference of the CT from the RSV protein F gene to the CT of β-actin gene. Fold-change in mRNA abundance was calculated using 2 (-ΔΔCT) method (Schmittgen and Livak, 2008) (link).