The Advanced Genome Technologies Core (AGTC) VGN Microarray Facility continually strives to provide comprehensive support for all projects requiring microarray approaches. The facility staff works collaboratively with both the VGN Bioinformatics Core and Molecular Bioinformatics Shared Resource teams to provide integrated project support from experimental design through data analysis. This past year the facility implemented two new genechips (Clariom S and Clariom D) including one with a new probe design (Clariom S). They also added the ability to request single sample qualitative assessments of nucleic acids, and completed a challenging sixty genechip project that required extensive workflow development and optimization.This assisted Dr. Sean Diehl, an assistant professor in the Department of Medicine-Infectious Diseases and associate director for research for the Vaccine Testing Center at the University of Vermont College of Medicine.
Dr. Diehl’s research project aims are to better understand the behavior of the immune system in malnourished children. The design for this study was to profile the transcriptomes and compare gender matched controls to samples from malnourished children in Bangledesh, using the Human Transcriptome Array that, by probe design, mimics a RNA-seq project. The project was presented to the microarray facility as whole blood mixed with Trizol reagent at incorrect ratios, and in some cases stored for extended periods at -80˚C. Testing was performed in the microarray facility to ascertain what method would provide the highest recovery of intact total RNA. While the NanoDrop spectrophotometer readings appeared to indicate sufficient recoveries, the target preparations were unsuccessful.
Suspecting insufficient total RNA loading into target preparations, the Qubit fluorometer was implemented. This led to successful target preparations combining a new workflow with small recovery samples quantified by fluorometric approaches. As a result, all new project samples require quantification via fluorometry. The project completion was advantaged by implementation of a new target preparation kit developed recently by Affymetrix called WT Pico kit that allows sample inputs as low as 170pg/ul. The data is currently being analyzed by the VGN Bioinformatics Core.
At a webinar presentation on October 4, 2016, Dr. Diehl presented the work from the facility optimizing his project workflow. The webinar discussed how novel transcriptome profiling assays can generate robust expression data to help identify RNA biomarkers from challenging samples such as feces and whole blood as well as explain the importance of choosing the optimal technology to obtain reliable data from rare samples thereby preserving material for future use.