Single Gonadotrope RNA Preservation and Transcriptional Analysis

Presentation Number: SUN 490
Date of Presentation: April 2nd, 2017

Frederique Murielle Ruf-Zamojski*1, Chirine Toufaily2, Yongchao Ge1, Venugopalan D Nair1, Judith L Turgeon3, German Nudelman1, Daniel J. Bernard2 and Stuart C. Sealfon1
1Icahn School of Medicine at Mount Sinai, New York, NY, 2McGill University, Montreal, QC, Canada, 3University of California Davis, Davis, CA

Abstract

Previous immunohistochemical and GFP-tagging studies of the gonadotrope have suggested that the anterior pituitary cells that mediate gonadotropin synthesis may actually comprise a complex and dynamic population of diverse cell types, such as somatotrope cells [1]. It is important, therefore, to characterize the global expression of individual transformed and primary gonadotropes and to study their transcriptional regulation at the single cell level of resolution.

Single cell transcriptomics has emerged as an important technique for refinement of cell subtype classification and for studying single cell response variation and gene co-regulatory modules. A major barrier for single cell transcriptomics studies is that cells must be maintained alive during the time interval between treatment or harvesting and the in vitro RNA transcription step, causing confounding gene expression changes to occur in response to cell manipulation. To overcome this limitation, we developed an RNA stabilization Buffer for Examination of Single cell Transcriptomes (RNA-Best [2]) that instantly fixes RNA expression, suspends cultured cells without trypsinization or handling, and preserves physically intact single cells for downstream analysis. We show that cells preserved in RNA-Best for 6 days at 4C give identical transcriptional results to freshly lysed cells. RNA-Best maintains single cell individuality for processing for single cell genomics using microfluidic chips. This method also allows cells studied in one laboratory to be transferred to another for single cell transcriptome analysis.

We applied this method to investigate LßT2 gonadotrope cell line single-cell gene regulation and single primary mouse pituitary cell gene expression using either integrated fluidic circuit multiplex real-time PCR or single-cell RNA sequencing. Our collaborators in Montreal stabilized primary pituitary cells in RNA-Best, shipped them on ice, and we later analyzed the samples in New York. Preliminary primary pituitary cell studies suggest a profound degree of heterogeneity of gonadotrope and mixed pituitary cell types. LßT2 cell regulation studies have identified novel candidate co-regulatory modules for controlling baseline levels of gonadotropin regulatory genes or the response to GnRH.

 

Nothing to Disclose: FMR, CT, YG, VDN, JLT, GN, DJB, SCS