Fifth Quarter Report – August 2018
Fifth Quarter Report – August 2018
This Project is a hypothesis-generating study for the control of lung damage in patients with idiopathic pulmonary fibrosis (IPF) and lung and skin damage in systemic scleroderma (SSc) patients both with and without interstitial lung disease (ILD). Enrolment of SSc patients with and without ILD to provide blood and skin tissue samples closed ahead of schedule during the first quarter by the end of August 2017 with samples stored in The Scleroderma Biobank at St. Paul’s Hospital in Vancouver. Enrolment of patients with idiopathic pulmonary fibrosis (IPF) and normal controls was completed by the middle of November. Skin fibroblast extraction and growth from skin samples (from all but the IPF patients from whom no skin samples were taken due to ethical reasons as their skin is not affected) were completed by the laboratory at St. Paul’s and stored in the Biobank before the Christmas 2017 break. Extra efforts were required by laboratory staff to harvest sufficient miRNA for two of the tissues, which doubled the time required for extraction but not for much-added cost in terms of purchasing reagents and other consumables. Extraction of miRNA from all five tissue types was completed by mid-April 2018 but miRNA extraction was not sufficient in the fifth tissue for whole genome sequencing (WGS) without additional processing by the laboratory and so this was placed on indefinite hold due to cost. A sixth tissue remains stored in the Biobank because its miRNA cannot be amplified for miRNA WGS, also for cost reasons, but will be analyzed by Nanostring miRNA expression chemistry.
The laboratory at St. Paul’s Hospital transferred 275 frozen tissue samples to the British Columbia Genome Sciences Centre (BCGSC) on May 16 2018, where these were entered in the queue for WGS analysis. On July 26 2018, processed miRNA sequence data from 92 duplicate samples of whole blood comprising 11 patients with both limited SSc and ILD, 19 patients with IPF, and 16 control participants was downloaded from BCGSC and quality control checks were commenced by the Project team. Raw miRNA sequence data for these samples were downloaded from BCGSC on August 7 2018 with quality control checking continuing until mid-August and then suspended due to vacations.
The original design called for miRNA WGS analysis on three tissue types and Nanostring miRNA expression analysis on four tissue types. This experimental design had to be altered because two more tissues were added and miRNA WGS analysis can only be done on three of the four original tissue types. Nanostring analysis requires one-tenth the amount of miRNA. There is ample miRNA for Nanostring analysis for five tissue types. In June, there was to be a developmental run using Nanostring for one tissue type from each of four patients with SSc-ILD, four patients with IPF, and four controls but this and WGS miRNA for the remaining 184 duplicate samples has been delayed until October pending completion of the quality control assessments.
University researchers involved in this project are Drs. James Dunne, Chris Ryerson, and Pearce Wilcox of The University of British Columbia and Dr. Kevin Keen of The University of Northern British Columbia.