Use of a Purpose-Built Impression Cytology Device for Gene Expression Quantification at the Ocular Surface Using Quantitative PCR and Droplet Digital PCR.

Abstract

To describe an impression cytology (IC) technique using a purpose-built, sterile, EYEPRIM IC device that can be coupled with a TRIzol reagent-based RNA extraction protocol to yield sufficient RNA for gene expression analysis. IC samples using the EYEPRIM device were collected from the bulbar conjunctiva, with and without topical anesthesia, and evaluated for RNA yield, the absence of polymerase chain reaction (PCR) inhibitors, and the ability to detect biomarkers by quantitative real-time PCR and droplet digital PCR. A technique for collecting IC samples in the clinic, while preserving RNA, and a protocol for subsequent laboratory analysis of RNA were developed. The extracted RNA was free of PCR inhibitors and could be synthesized into complementary DNA and used for successful relative quantification of ocular surface biomarkers by quantitative real-time PCR. For gene targets present in low abundance, complementary DNA could also be used for quantification by the relatively new and emerging method of droplet digital PCR. The described method was successfully used to evaluate 3 biomarkers in a clinical trial assessing the tolerability of a proprietary eyelid therapy in 92 IC samples from a study population of 46 participants. IC is a recognized technique for ocular surface cell evaluation and protein biomarker quantification but is infrequently used for quantifying gene expression. The EYEPRIM device allows ease of use and impression-to-impression consistency while accurate gene expression data offers a highly specific and sensitive method of disease characterization for clinician scientists to use in diagnosis.