A rapid method for gene expression analysis of Borna disease virus in neurons and astrocytes using laser microdissection and real-time RT-PCR.

Abstract

Laser microdissection combined with real-time RT-PCR represents a powerful method to analyse the transcription efficiency of defined cell types. Therefore, a RNA-preserving immunolabelling method was established to identify neurons and astrocytes in persistently BDV-infected rat brain sections for subsequent laser microdissection and quantitation of viral gene products by real-time RT-PCR. Firstly, to ensure an accurate measurement of viral RNA after immunolabelling, different reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], succinate-ubiquinone reductase [SDHA], hypoxanthine phosphoribosyl-transferase-1 [HPRT]) were tested. Only normalisation with GAPDH yielded a stable relative expression of viral RNA encoding the nucleoprotein (BDV-N), the matrixprotein and the glycoprotein (intron I and intron II). The two remaining reference genes biased the ratios of BDV-transcripts in the immunolabelled brain sections significantly. Secondly, 100 immunolabelled neurons and astrocytes were harvested using laser microdissection and amplification of all viral transcripts revealed 681 and 168 (BDV-N), 573 and 254 (intron I), 324 and 133 (intron II) and 161 and 36 (GAPDH) absolute copy numbers in neurons and astrocytes, respectively. Thus, laser microdissection combined with real-time RT-PCR provides an effective tool for the analysis of cell-specific viral transcription efficiency and allows elucidating virus-host-interactions and virus persistence mechanisms in the CNS.