Online Data Supplement

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1 Persistence of Rhinovirus Infection of Lower Airways in Patients with Bronchial Asthma Monika Wo, Marek Sanak, Jerzy Soja, Henryk Olechnowicz,William W. Busse, Andrew Szczeklik Online Data Supplement 39

2 Probe.pdf Sequence of a 536 base pairs HRV probe cloned (clone 18mf.ab1, sequenced on ABI 377, Applied Biosystems) from an acutely infected nonasthmatic subject and used for hybridization following labeling with digoxygenin. The probe localizes in VP2/VP4 coding region of HRV genome and has at least 79% identity to phylogenetically different 55 HRV type A serotypes. Primers sequences are highlighted within the probe. IS-RTPCR.doc Additional data on the probe isolation, synthesis and indirect in situ RT-PCR hybridization Nasal lavages A nonasthmatic individual demonstrating acute common cold served as a source of HRV material. Each nostril was washed using 5 ml of isotonic natrium chloride. Cell suspension (200 Wl) was lysed using TRIzol reagent (Invitrogen) and RNA precipitated according to the manufacturer recommendations. Hybridization probe synthesis Reverse transcription (RT) was performed using AMV reverse transcriptase (Amresco) and universal reverse primer (5 - GGT AGT TTC CAC CAC CAA CC), selected from VirOligo database ( OL86-1) and modified by removal of degenrated nucleotides; 6Y was replaced with T and 18N with A, in addition adenine to guanine was changed at nucleotide 5 to strengthen primer homology to HRV 2 and HRV 16 sequence. The reaction was performed as recommended by manufacturer with temperatures: 60 minutes at 42 C 40

3 and 15 minutes at 75 C. Product of reverse transcription reaction was amplified using PCR method by addition of the universal forward primer (5 - CTA CTT TGG GTG TCC G, OL24). The reaction temperatures were as follows: 2 min 94 C, 38 cycles each of 45 sec at 94 C, 45 sec at 55 C, 1 mi at 72 C. The final extension was at 72 C for 10 min. Product of amplification was separated by 1,5% agarose gel electrophoresis, purified using the Gel Extraction Kit (Qiagex, USA), and cloned into TOPO plasmid vector using the TOPO TA Cloning Kit (Invitrogen, USA) according to the manufacturer recommendations. After identification of the insert by a direct sequencing (BigDye v.3.1 kit, Applied Biosystems), a probe was generated from the plasmid template by a direct labeling with digoxygenin (Dig Labelling Mix, Boehringer Ingelheim, Germany) during PCR, as recommended by the manufacturer, with the same primers as for cloning. Digoxigenine-labeled cdna was purified using Gel Extraction Kit (Qiagex, USA) following agarose gel separation and used, after denaturation, for detection of HRV specific in situ amplified material. Indirect in situ RT-PCR hybridization Formaldehyde fixed (4%) and paraffin embedded biopsies were sectioned into 4Wm thick slides and mounted on In Situ PCR Glass Slides (Perkin Elmer, USA). All the steps preceding reverse transcription had been completed in a RNase-free environment. Sections were dewaxed, rehydrated and digested with proteinase K solution (4 µg/ml, Promega, USA), for 5 minutes. Reverse transcription was performed using the reverse primer and AMV reverse transcriptase (Amresco, USA) according to the manufacturer protocol. The reaction was carried out with the following program: 60 minutes at 65 C and 15 min at 75 C. Samples overlaid with the reaction mixture we covered with AmpliCover Discs and secured with AmpliCover Clips (Perkin 41

4 Elmer, USA), then reverse transcribed in thermal cycler GeneAmp In Situ PCR System 1000 (Perkin Elmer, USA). Thereafter reverse transcription mixture was replaced with PCR mixture, containing Taq polymerase (Finnzymes, Finland), the two primers and each triphosphonucleotide. Thermal protocol included initial denaturation step (92 C for 1 minute) followed by 15 cycles of denaturation at 94 C for 2 minutes, annealing at 54 C for 2 minutes and elongation at 72 C for 4 minutes. The reaction was completed by two brief sequential washes in 2x standard saline citrate (SSC). Digoxygenine labeled and heat-denaturated probe (250 ng/ml) was hybridized overnight at 42 C. Post hybridization washes included 2 x SSC for 30 minutes and 0.2 x SSC for 15 minutes at 55 C. The probe attached to amplified cdna was detected by immunohistochemistry using anti-dig Fab fragments conjugated with alkaline phosphatase (Boehringer Ingelheim, Germany) and 5-bromo-4-chloro-3-indolyl-phosphate substrate (BCIP) according to the manufacturer protocol. Color precipitate developed during 16-hour incubation. 42

5 Figure E1. 43