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1Department of Molecular and Microbiology, George Mason University, 2Krasnow Institute for Advanced Study, George Mason University
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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.
Keywords: JoVE Neuroscience, Issue 39, Drosophila, neuron, Peripheral Nervous System (PNS), dendritic arborization neurons, RNA, microarray, qRT-PCR, cell isolation, Laser Capture Microdissection (LCM)
Iyer, E. P. R., Cox, D. N. Laser Capture Microdissection of Drosophila Peripheral Neurons. J. Vis. Exp. (39), e2016, doi:10.3791/2016 (2010).
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 morphogenesis1,2. 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 purification3,4, 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 type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, 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.
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
If it is necessary to preserve the tissue morphology for identifying the cells of interest, or if the expected number of cells per section is found to be satisfactory then proceed directly to Step 11.
Alternatively if the cells are labeled specifically and can be identified with an easily identifiable marker such as GFP or RFP, but the number of cells per section is found to be low, then Steps 5-10 may be helpful for increasing the number of cells available for capture per section. These are optional steps and should be considered only if required, as a method for increasing the number of da neurons available for LCM per section when the other method fails to provide favorable results. [Caution: This method may disrupt the overall tissue morphology and make tissue identification based on specific spatial location very difficult.]
2. Dehydration and Cuticle Removal from Frozen Larval Sections
[Critical Step: All solutions for dehydration must be prepared fresh before each LCM session. Complete dehydration of frozen larval tissue sections is essential for achieving optimal microdissection efficiency]
|70% Ethanol (Fixative)||3-10 Seconds|
|2.5% Trypsin Incubation||5-30 Seconds|
|70% Ethanol||60 Seconds|
|95% Ethanol||60 Seconds|
|100% Ethanol||120 Seconds|
|100% Ethanol||120 Seconds|
|100% Xylene||120 Seconds|
|100% Xylene||120 Seconds|
|Air dry in a mild air stream||60-120 Seconds|
Table 1: Recommended procedure for LCM dehydration of frozen larval tissue sections.
3. Laser Capture Microdissection
PixCell IIe LCM instrument equipped with Fluor 300 epifluorescence optics optimized for EGFP was used for performing the LCM.
[Critical Step: If possible, perform the microdissection in a humidity-controlled room to prevent any reduction in microdissection efficiency due to increased humidity. Room humidity is a critical factor affecting the microdissection efficiency. Low room humidity can cause an increased static-cling resulting in non-specific capture, while high room humidity can result in low microdissection efficiency. We achieved optimal LCM efficiency between 25-50% relative humidity conditions.]
4. RNA isolation from LCM derived cells
Laser capture microdissection (LCM) was used to isolate single, class-specific or multiple Drosophila da neurons from third instar larvae (Figure 1). LCM allows for a high degree of precision and specificity in the isolation of Drosophila da neuron cell bodies (Figure 2a-c). Moreover, the total RNA purified from these isolated da neurons was found to be of excellent quality as indicated by the presence of sharp 5.8S, 18S and 28S ribosomal RNA peaks when analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) (Figure 2d).
To assess the neuronal-specific enrichment of our isolated cells we performed real-time quantitative reverse transcription PCR (qRT-PCR) using the neuronal gene-specific marker, elav. For the qRT-PCR analyses, we calculated the relative levels of elav expression in the LCM microdissected da neurons and normalized this expression to that of our endogenous control (rp49) and relative to total RNA isolated from whole larvae using the ΔΔCt method23. These analyses revealed over 2.5 fold enrichment in the relative levels of elav in LCM micro-dissected da neuron samples as compared to whole animals (Figure 3).
Figure 1: LCM of Drosophila peripheral neurons. (a) Age matched third instar larvae are selected, washed and (b) embedded in a cryomold with OCT and frozen at -80°C, (c) 8μm serial tissue sections are created using a standard cryostat and (d) placed uniformly on clean glass slides. The glass slides with the affixed tissue sections are stored at -80°C prior to LCM processing. (e,f) Immediately preceding LCM, the tissue sections are thawed and briefly fixed in 70% ethanol followed by brief rinse in ddH2O. (g) The slides are briefly incubated with trypsin and rinsed with ddH2O to remove cuticles (black) from the larval sections. (h,i) tissue sections without larval cuticle are then further dehydrated in an ethanol gradient and finally cleared in xylene. (j) The LCM cap with a thermolabile polymer is placed on the tissue section and the laser is pulsed on the selected fluorescent cell body. The laser pulse melts the polymer and engulfs the selected cell body. The polymer cap, along with the captured cell body is lifted, and (k) RNA extraction buffer is added to the cell. The captured cell, along with the RNA extraction buffer is incubated at 42°C for 30 minutes and can be either stored at -80°C, or directly processed for RNA purification.
Figure 2: LCM facilitates high precision capture of da neurons. (a) Representative image of a dehydrated and trypsin treated 8 micron tissue section prior to performing LCM, showing two class-IV da neurons labeled with GFP by the ppk-GAL4,UASmCD8::GFP reporter strain. Note, one of the neurons is highlighted (arrowhead) for capture. (b) Cell body of the highlighted neuron (arrowhead) is cleanly micro-dissected with high specificity from the tissue section. (c) End-on view of a single class-IV da neuron captured on the LCM cap. (d) Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) electropherogram of total RNA isolated from LCM-derived da neurons, showing excellent RNA quality as indicated by the presence of sharp 5.8S, 18S, and 28S rRNA peaks.
Figure 3: qRT-PCR reveals substantial enrichment of neuronal marker gene expression in LCM captured da neurons relative to whole animal. qRT-PCR analyses of neuron-specific marker gene expression (elav) in LCM captured da neurons (21-7-GAL4,UAS-mCD8::GFP) and whole animals in age matched third instar larvae was performed in triplicate. Relative levels of elav expression in LCM captured da neurons were normalized to the endogenous control (rp49) and relative to whole larvae using the ΔΔCt method23. A 2.5 fold enrichment in the relative levels of elav in LCM captured da neuron samples was observed relative to whole animals.
Problem: RNA degraded, or of low quality.
Ensure RNAse free condition throughout experiment. Try reducing amount of time spent on LCM per slide to 30 minutes. Keep the slides and samples on dry ice except when performing LCM. Avoid thawing the tissue sections once they are cut.
Problem: Most cells are lost from the slide during trypsin treatment (step 17-18).
Try reducing the time of trypsin treatment. Try reducing the trypsin concentration.
Problem: Presence of cuticle and other non-specific tissue debris on the cap polymer.
Try removing the tissue debris by blotting the polymer surface with the tacky side of an adhesive note22.
Problem: Low LCM efficiency.
Try increasing the incubation time in 100% ethanol and xylenes. Reduce room humidity using dehumidifiers.
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 which label the cell type or types of interest. Our results demonstrate that the RNA obtained from laser capture micro-dissected da neurons is of very high quality allowing for potential direct use in qRT-PCR or microarray-based analyses. Applications of this protocol extend beyond the capture of wild-type cells of interest, as discussed here, where LCM could be used in combination with loss-of-function (UAS-RNAi) and/or gain-of-function studies employing the GAL4/UAS system in a cell type of interest.
No conflicts of interest declared.
We thank Drs. Yuh-Nung Jan and Wes Grueber for providing fly stocks used in this study, and Virginia Espina, Dr. Emanuel Petricoin and Dr. Lance Liotta for assistance with LCM. The authors acknowledge the Thomas F. and Kate Miller Jeffress Memorial Trust for support of this research (D.N.C.) and the George Mason University Provost s Office (E.P.R.I.).
|10X Phosphate Buffered Saline (PBS)||MP Biomedicals||PBS10X02||Diluted to 1X working solution|
|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|
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