Lung tissue analysis for RSV titre
A portion of lung tissue was snap frozen in liquid nitrogen for analysis
of viral load by real-time quantitative PCR (qPCR). RNA lysis buffer was
added to frozen lung tissue and homogenised using a microfuge pestle on
ice. Samples were centrifuged at 11 000 rpm for 5 mins and total RNA
extracted from resulting supernatants using Purelink RNA extraction kit
(Ambion, New Haven, USA). A minimum of 2 mg used as a template for cDNA
synthesis using random primers and M-MLV reverse transcriptase (Promega,
Southampton, UK). The presence of RSV was measured using a commercially
available TaqMan real-time PCR kit designed to determine RSV titre by
interpolation to a standard curve (RSVSpp, GeneSig, Southampton, UK).
The PCR reaction was analysed using Applied Biosystems ABI 5400
thermocycler.