Immuno-guided laser-capture microdissection of glial cells for mRNA analysis

Abstract

Laser-capture microdissection (LCM) allows for retrieval of specific cell populations in situ. By combining immunofluorescent labeling with LCM, mRNAs can be probed by qRT-PCR for determining in situ gene expression during health and disease. This approach permits obtaining and analyzing histologically enriched cell populations in a tissue that can be hardly obtained from other methods such as white matter astrocytes from rodents or any individual cell population from archival human or rodent brain tissues. Herein, we present our methodology of laser-captured mouse spinal cord white matter astrocytes, which can be adapted for any cell type in CNS tissue and low RNAse containing tissues. The methods presented with an emphasis on tips and advices include the cryostat section preparation from snap-frozen tissue, an adapted immunofluorescent labeling, a brief overview of LCM using a UV-based technology with polyethylene membrane glass slides, procedures for direct use of RNA from lysis buffer vs. column-based purified RNA, RNA quality/quantity assessment, the reverse transcription and preamplification steps used before real-time qPCR analysis.