Laser Capture Microdissection of Drosophila Peripheral Neurons

Summary

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Abstract

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis^1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification^3,4, Fluorescent Activated Cell Sorting (FACS)^5-8, and RNA binding protein based strategies⁹, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type^10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells^17,18, including da neurons at the third instar larval stage of development.

Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP¹⁹ transgene under the control of either the class IV da neuron specific ppk-GAL4²⁰ driver or the pan-da neuron specific 21-7-GAL4²¹ driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Protocol

General Comments on LCM of Drosophila Peripheral Neurons

Allow from 6 hours, up to a week or longer for LCM depending upon the tissue type and the number of cells required.

All the procedures are carried out in strictly RNAse-free conditions following standard procedures. Larvae expressing either 21-7-GAL4,UAS-mCD8::GFP or ppk-GAL4,UAS-mCD8::GFP transgenic reporter lines were used for these experiments.

1. Preparing the larvae

1. Collect 30-40 age-matched third instar larvae and wash them in ddH₂O followed by brief rinses in RNAse Away (Si....

Discussion

The protocol presented herein describes our optimized method for the isolation of Drosophila peripheral neurons via LCM. While this LCM protocol was designed for the specific isolation of single, class-specific or multiple Drosophila da neurons from the third instar larval stage of development, minor modifications of the protocol could readily be adapted for the capture of other Drosophila cell types from all developmental stages using distinct GAL4,UAS-mCD8-GFP reporter transgenes wh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X Phosphate Buffered Saline (PBS)	MP Biomedicals	PBS10X02	Diluted to 1X working solution
2.5% Trypsin	Sigma-Aldrich	T1426
RNase-AWAY	Sigma-Aldrich	83931
Xylenes, histological grade	Fisher Scientific	X3S-4
RNAse-free water	Fisher Scientific	BP561-1
Optimal Cutting Temperature (OCT) compound	Tissue-Tek	4583
PicoPure RNA Isolation Kit	Molecular Devices	KIT0204	Follow manufacturer’s instructions
Thin walled reaction tube with domed cap	Applied Biosystems	N8010611
ExtracSure Sample Extraction Device	Molecular Devices	LCM 0208
Incubation Block for sample extraction from CapSure HS Caps	Molecular Devices	LCM0505
Alignment Tray for CapSure HS LCM Caps and ExtracSure Sample Extraction Devices	Molecular Devices	LCM0504
CapSure HS LCM caps	Molecular Devices	LCM0213
75x100 mm glass slides	Fisher Scientific	12-544-3
Tissue embedding molds	Fisher Scientific	NC9642669
Polypropylene pestle for 1.5 ml microcentrifuge tubes	USA Scientific, Inc.	1415-5390
70% Ethanol; 95% Ethanol; 100% Ethanol
Dry ice
Equipment: Cryostat 50 ml conical tube for slide fixation, rinsing, trypsin treatment and ethanol/xylene dehydration -80°C freezer Incubator PixCell IIe LCM Instrument with Fluor 300 epifluorescence optics optimized for EGFP (Molecular Devices-Molecular Devices)