Разработка нового метода детектирования экспрессии генов с помощью количественной ОТ-ПЦР единичных клеток без выделения РНК в сочетании с лазерной захватывающей микродиссекцией

Abstract

Laser capture microdissection (LCM) combined with real time RT-PCR represents a powerful method for analyzing gene expression levels in selected cell types. To avoid degradation of RNA by endogenous and exogenous RNases and to ensure selection of target cells only, we designed a protocol in which the inactivation of exogenous and endogenous RNases by RNaseZap and RNA later is combined with immunofluorescent labeling, and labeled cells in sections used for subsequent LCM and real time RT-PCR. Immunolabelled neurons were captured onto the caps of RNase-free microcentrifuge tubes using LCM and lysed with 3% NP-40 without prior RNA purification. Subsequent reverse transcription for cDNA synthesis was performed in situ on the cap surface to avoid mRNA loss due to transfer between tubes. Applying this protocol, we determined immunoglobulin G (IgG), nerve growth factor (NGF) and GAPDH mRNA levels into small numbers of captured neurons with real time RT-PCR. Thus, this novel method combining LCM with real time RT-PCR without RNA purification appears to be an effective tool for the analysis of cell-specific target gene transcription in small numbers of cells isolated from RNA later-treated tissues.