Adaptation of Impression Cytology to Enable Conjunctival Surface Cell Transcriptome Analysis

Abstract

Purpose: This study investigates the extent of the human transcriptome that can be quantified from conjunctival impression cytology extracts. The aim is to determine if sufficient RNA can be isolated from a patient’s conjunctival surface to identify differences in gene expression between dry eye and normal patients of (a) an array of 96 inflammatory biomarkers and associated receptors, and (b) if this comparison can be expanded to the entire transcriptome.

Materials and Methods: CIC was used to collect conjunctival surface cells from 53 qualifying normal and dry eye patients. Based on prior optimization of all assay steps, RNA was isolated from the samples using a Qiagen RNeasy Plus Mini Kit and qRT-PCR was used to determine gene expression of 96 genes using TaqMan Low Density Array cards. Samples from six normal and six dry eye patients were then assayed on an Illumina Human HT-12 BeadChip.

Results: Optimization steps yielded an RNA processing procedure that improved yield from an initial 12 genes through 96, then to the entire human transcriptome. For the HT-12 BeadChip, more than 30 genes differed by a factor of >1.5 between the dry eye and normal groups and seven genes were down-regulated by a factor of >2.0 in the dry eye group: HLA-DRB5, PSCA, FOS, lysozyme, TSC22D1, CAPN13 and CXCL6.

Conclusions: Conjunctival impression cytology can be used to collect sufficient RNA from conjunctival surface cells that, when processed optimally, allows successful transcriptome-wide expression analysis. While the current transcriptome analysis used a limited patient group, larger studies of patients with various types and severities of dry eye should reveal significant gene expression trends that can then be targeted to improve dry eye treatment options.

• Biomarkers
• conjunctival impression cytology
• dry eye
• gene expression
• transcriptome

Acknowledgements

Thanks to Dr Michael Frost for a critical review of the article and to the UAB Heflin Center for Genomic Sciences for running the Illumina Human HT-12 BeadChip.