Name: a facs-based protocol to isolate rna from the secondary cells of drosophila male accessory glands
Uploaded: 2019-09-05T15:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands at JoVE.com

We are grateful to the members of the Karch lab, to the iGE3 genomics plateform, to the Flow Cytometry core facility of the University of Geneva, and to Dr. Jean-Pierre Aubry-Lachainaye who set the protocol for FACS. We thank Luca Stickley for his help with visualizing the reads on IGV. We thank ourselves for gentle permission to reuse figures, and the editors for prompting us to be creative with writing.
This research was funded by the State of Geneva (CI, RKM, FK), the Swiss National Fund for Research (www.snf.ch) (FK and RKM) and donations from the Claraz Foundation (FK).

1. Drosophila Lines Used and Collection of Males
<ol>
	<li>For this protocol, use males expressing GFP in the SCs and not the main cells. Here, an AbdB-GAL4 driver (described in reference18), recombined with UAS-GFP (on chromosome 2) is used. Other appropriate drivers can also be used. For isolation of SCs under different mutant conditions, cross the mutation into lines containing the SC-specific GFP line.</li>
	<li>Collect two batches of about 25 healthy, virgin males for each genotype and age them at 25 &#176;C for 3-4 days in vials with freshly made Drosophila food. 
	NOTE: As age, mating status, nutrition and social environment each affect reproductive biology, strict protocols must be followed to control for these parameters. Virgin males are aged for 3-4 days after pupal eclosion at 25 &#176;C with a 12 h/12 h light/dark cycle, on standard corn meal-agar-yeast food, in groups of 20-25 males per vial.</li>
</ol>
2. Solutions and Material Preparation
<ol>
	<li>Prepare the following solutions.
	<ol>
		<li>Prepare Serum Supplemented Medium (SSM): Schneider&#39;s Drosophila medium complemented with 10% heat inactivated fetal bovine serum and 1% Penicillin-Streptomycin.</li>
		<li>Prepare aliquots of 1x trypsin enzyme (e.g., TrypLE Express Enzyme) and store at room temperature.</li>
		<li>Prepare aliquots of papain [50 U/mL] (stored at -20 &#176;C and thawed only once).</li>
		<li>Prepare 1x phosphate buffer saline (PBS, stored at room temperature).</li>
	</ol>
	</li>
	<li>Prepare multiple flame-rounded pipet tips for the physical dissociation of accessory glands. 
	NOTE: Use low retention tips for handling accessory glands as they tend to adhere to untreated plastic. The process of flame-rounding reduces the tip opening and smoothens the edges of the tip. This ensures an efficient dissociation after peptidase digestion without shearing the cell membranes.
	<ol>
		<li>Cut one 200 &#181;L tip with a sharp blade and gently pass it over a flame to round out its tip so that the opening is wider but smooth for handling during step 3.7.</li>
		<li>Narrow the opening of multiple 1,000 &#181;L tips by passing the tip opening near a flame for less than one second. Rotate the tip slightly to avoid over-melting or clogging. Sort the tips made from narrower to wider. This can be done by timing the speed of aspiration. Try to dissociate a small scale sample of AGs to test the efficiency of the narrowest tips. 
		NOTE: These tips are critical for mechanical agitation (steps 5.2 and 5.3). Quality flame-rounded pipet tips allow for complete dissociation while preserving cell viability. As such, these tips are washed thoroughly with water at the end of the procedure for reuse. This allows for day to day reproducible dissociation.</li>
	</ol>
	</li>
</ol>
3. Dissection of Accessory Glands
<ol>
	<li>Put 20 to 25 male Drosophila in a glass dish on ice.</li>
	<li>Dissect 1 male in SSM. Take off its reproductive tract and clear the accessory gland pair from all other tissues apart from the ejaculatory duct. 
	NOTE: Remove testes because the released sperm can create clumps and disturb the dissociation process. Remove the ejaculatory bulb, which makes handling difficult when it floats.</li>
	<li>With forceps, transfer accessory glands to a glass plate filled with SSM at room temperature. Repeat steps 3.2-3.3 20 times to obtain a batch of 20 pairs of glands in SSM. 
	NOTE: These steps will be achieved in 15 to 20 min. AGs should look healthy, and the peristaltic movements of the muscle layer around glands should be visible. GFP can be monitored using a fluorescent microscope.</li>
	<li>Transfer accessory glands to 1x PBS for a 1-2 min wash at room temperature. 
	NOTE: For better dissociation, it is imperative to rinse the glands with PBS. The duration of this step, however, should be limited as the cells show signs of stress in PBS; dissociated secondary cells in PBS rapidly swell and die.</li>
	<li>Dilute 20 &#181;L papain [50 U/mL] into 180 &#181;L of 1x trypsin enzyme to obtain the dissociation solution (to scale up or down keep 9 &#181;L of trypsin enzyme and 1 &#181;L of papain for each male). With forceps, transfer accessory glands to this solution.</li>
	<li>Isolate the tip of accessory glands (containing secondary cells) from the proximal part. Use fine forceps to pinch firmly the middle of a glandular lobe and cut with the sharp tip of a second forceps. Remove the proximal part of accessory glands and the ejaculatory duct to improve dissociation and reduce cell sorting time. 
	NOTE: Dissecting the distal part from 20 pairs of glands will take 15 to 20 min and digestion by peptidases will thus start at room temperature.</li>
	<li>After all gland tips have been dissected, transfer them into a 1.5 mL tube using a special 200 &#181;L pipet tip prepared at Step 2.2.1. It should be wide, rounded and wet prior to handling glands. 
	NOTE: To pipet accessory gland tissue, tips should always be pre-wet with appropriate solution (trypsin enzyme or SSM, keep one tube of each for this purpose). Carefully rinse tips between samples to avoid contamination.</li>
</ol>
4. Tissue Digestion
<ol>
	<li>Place the microtube in a 37 &#176;C shaker for 60 min, rocking at 1,000 rpm. 
	NOTE: Both agitation and digestion time are critical for successful dissociation. Shorter or motionless digestion will give poor dissociation, probably because the outer muscle layer and inner viscous seminal fluid protect accessory gland cells from peptidases.</li>
	<li>Add 1 mL of SSM at room temperature to stop the digestion and proceed immediately to step 5.</li>
</ol>
5. Mechanical Dissociation of the Cells
<ol>
	<li>Using a wide rounded 1,000 &#181;L tip, pre-wet with SSM, transfer the sample to a 24-well plate. Monitor GFP fluorescence under the microscope: most gland tips should look intact and a few SC should be detached.</li>
	<li>Using a rounded narrow pipet tip generated at Step 2.2.2, pipet up and down 3 to 5 times to disrupt the accessory gland tissue. 
	NOTE: Monitor fluorescence to make sure that big tissue patches have been broken up. Repeat step 5.2 if this is not the case.</li>
	<li>Using a very narrow rounded pipet tip (generated at Step 2.2.2), pipet up and down 1-2 times. 
	NOTE: Only individual cells should be visible after Step 5.3. Using 24-well plates is recommended because they enable an easy monitoring of the process. When a few samples were processed and gave satisfactory result (healthy looking secondary cells perfectly dissociated), the pipetting steps will be performed in the microtube.</li>
	<li>Wait at least 15 min to let the cells settle at the bottom of the well and remove the excess SSM to reduce the sorting time. 
	NOTE: Letting cells settle proved superior to alternative methods like centrifugation, and allows a last visual inspection of cells just before FACS.</li>
	<li>Pipet the cells into a clean 1.5 mL tube and pool identical samples (two batches of 20 males for each condition). Rinse wells with SSM to recover last cells, and pipet them into the tube (use a small volume).</li>
	<li>In 1.5 mL microtubes, add 300 &#181;L of Cell Lysis Solution and 1 &#181;L of Proteinase K [50 &#181;g/&#181;L] (solutions provided in the RNA purification kit, see Step 7). Prepare two tubes for each sample (one for MCs and one for SCs).</li>
</ol>
6. FACS (Fluorescence-activated Cell Sorting)
<ol>
	<li>Add viability marker to each sample to be sorted a few minutes before sorting (0.3 mM Draq7). 
	CAUTION: Draq7 should be handled with caution.</li>
	<li>Sort a homogeneous population of live secondary cells (GFP-positive, Draq7-negative cells). In another microtube, sort a population of main cells (smaller, GFP-negative, Draq7-negative cells). Use the following FACS gating strategy: 
	NOTE: Set the cell sorter pressure at 25 PSI and pass cells through a 100 &#181;m nozzle. The sorting rate is around 2,000 cells/s.
	<ol>
		<li>Exclude debris and select total cells based on FSC-SSC (Figure 2E) in order to exclude debris.</li>
		<li>Exclude dead cells. Excite Draq7 with a 640 nm laser and collect emitted fluorescence with a 795/70 band-pass filter. Gate Draq7-positive cells out (Figure 2F).</li>
		<li>Remove doublets using a double gating on SSC-H vs SSC-W and FSC-A vs FSC-H (Figure 2H). 
		NOTE: Exclude cell doublets stringently using double gating, to reduce main cell contamination.</li>
		<li>Sort ~550 GFP-positive cells into a 1.5 mL microtube with Lysis buffer and Proteinase K. This population is considered secondary cells (SC, Figure 2G). Excite GFP at 488 nm and collect emitted fluorescence with a 526/52 band-pass filter.</li>
		<li>Sort ~1,000 GFP-negative cells, homogeneous and small in size, into a 1.5 mL microtube with Lysis buffer and Proteinase K. This population is considered main cells (MC, Figure 2G).</li>
		<li>Vortex all samples. 
		NOTE: From 40 males (~40 x 80 SC = 3,200 secondary cells), 600 to 800 live singlet secondary cells are obtained (around 20-25% retrieval). Stop sorting around 550 SC and 1,000 MC to normalize samples.</li>
	</ol>
	</li>
</ol>
7. RNA Extraction
NOTE: We used Epicentre MasterPure RNA Purification Kit for RNA extraction, with the following adaptations. Other kits might be used as long as the yield is high enough to prepare a library for sequencing from ~500 cells (2 ng RNAs was used here).
<ol>
	<li>Heat samples at 65 &#176;C for 15 min and vortex every 5 min to complete cell lysis.</li>
	<li>Place samples on ice for 5 min. Follow manufacturer&#39;s recommendations for nucleic acids precipitations (parts &#34;Precipitation of Total Nucleic Acids&#34; and &#34;Removal of Contaminating DNA from Total Nucleic Acid Preparations&#34;).</li>
	<li>Suspend RNA pellet in 10 &#181;L of RNase-free TE buffer.</li>
	<li>Add RNase inhibitor (optional).</li>
	<li>Store samples at -80 &#176;C.</li>
</ol>
8. Quality Controls of RNA Quantity, Quality and Specificity
<ol>
	<li>Estimate RNA quality and concentration. Due to the small volume and concentration, use RNA 6000 Pico chips here. Good quality RNA is defined as non-degraded, visible as a low baseline with sharp peaks corresponding to rRNAs.</li>
	<li>RT-qPCR to control the identity of sorted cells
	<ol>
		<li>Perform reverse transcription with 2 ng of total RNAs using random hexamers as primers. Perform RT on secondary cell RNA and main cell RNA. 
		NOTE: cDNAs can be diluted, aliquoted, and kept frozen for later use.</li>
		<li>Perform real time quantitative PCR using appropriate primer pairs to quantify housekeeping genes (alpha-Tubulin, 18S rRNA), secondary cell specific genes (MSA, Rab19, Abd-B) and main cell specific gene (Sex peptide).</li>
	</ol>
	</li>
</ol>
9. Sequencing (cDNA Library Preparation, Sequencing and Data Analysis)
<ol>
	<li>Use 2 ng of total RNAs to synthesize cDNAs with polydT primers. Use SMARTer technology to amplify them for amplification.</li>
	<li>Use a Nextera XT kit to prepare the library.</li>
	<li>Sequence using multiplexed, 100 nucleotides single reads sequencing (even though 50 nucleotides reads are appropriate for most purposes) to yield around 30 million reads for each sample.</li>
</ol>
10. Data Analysis
<ol>
	<li>Run FastQC.</li>
	<li>Use the STAR aligner to map the reads on the UCSC dm6 Drosophila reference genome and generate .bam files. Use Integrative Genomics Viewer (IGV) to visualize reads on the genome browser.</li>
	<li>Use HTSeq to perform gene counts.</li>
	<li>Use the Trimmed Mean of M-values (TMM) method to normalize gene counts22. Use edgeR to perform statistical analysis of differential expression, MA plots and PCA.</li>
	<li>For gene expression analysis and statistics, use General Linear Model, quasi-likelihood F-test with False Detection Rate (FDR) and Benjamini &#38; Hochberg correction (BH).</li>
</ol>

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The authors have no disclosure.

Methods for cell dissociation from Drosophila tissue like imaginal discs are already described23. Our attempts to simply use these procedures on accessory glands failed, encouraging us to develop this new protocol. Protease digestion and mechanical trituration were critical steps we troubleshot for the success of the procedure, and we thus placed many notes in sections 2 to 5 to help experimenters to obtain satisfactory results. For dissociation to be successful, peptidases must reach the...

Organs are made up of multiple cell types, each with discrete functions and sometimes expressing vastly different sets of genes. To get a precise understanding of how an organ functions, it is often critical to study each distinct cell type that makes up this organ. One of the primary methods used to explore possible function is transcriptome analysis. This powerful method provides a snapshot of the gene expression in a cell, to reveal active processes and pathways. However, this type of analysis is often difficult for rare cell populations that must be purified from far more-abundant neighboring cells. For example, the Drosophila male accessory gland is an organ made up primarily of two secretory cell types. As the rarer of the two cell types comprises only 4% of the cells of this gland, the use of a cell-type specific transcriptome analysis had not been used to help determine the function of these cells.
Accessory glands (AGs) are organs of the male reproductive tract in insects. They are responsible for the production of most of the proteins of the seminal fluid (seminal fluid proteins (SFPs) and accessory gland proteins (ACPs)). Some of these SFPs are known to induce the physiological and behavioral responses in mated females, commonly called the post-mating response (PMR). Some of the PMRs include: an increased ovulation and egg-laying rate, the storage and release of sperm, a change in female diet, and a decrease in female receptivity to secondary courting males1,2. As insects impact many major societal issues from human health (as vectors for deadly diseases) to agriculture (insects can be pests, yet are critical for pollination and soil quality), understanding insect reproduction is an important area of research. The study of AGs and ACPs has been advanced significantly with the model organism Drosophila melanogaster. These studies have highlighted the role of AGs and some of the individual proteins that they produce in creating the PMR, impacting the work in other species like the disease vector Aedes aegypti3,4, and other insects1,5. Furthermore, as AGs secrete the constituents of the seminal fluid1,6 they are often thought of as the functional analog of the mammalian prostate gland and seminal vesicle. This function similarity combined with molecular similarities between the two tissue-types, have made the AGs a model for the prostate gland in flies7.
Within the Drosophila male, there are two lobes of accessory glands. Each lobe can be seen as a sac-like structure made up of a monolayer of secretory cells surrounding a central lumen, and wrapped by smooth muscles. As mentioned above, there are two morphologically, developmentally and functionally distinct secretory cell types making up this gland: the polygonal-shaped main cells (making up ~96% of the cells), and the larger, round secondary cells (SC) (making up the remaining 4% of cells, or about 40 cells per lobe). It has been shown that both cell types produce distinct sets of ACPs to induce and maintain the PMR. Most of the data obtained to date highlight the role of a single protein in triggering most of the characteristic behaviors of the PMR. This protein, the sex peptide, is a small, 36 amino acid peptide that is secreted by the main cells8,9,10. Although the sex peptide seems to play a major role in the PMR, other ACPs, produced by both the main and secondary cells, have also been shown to affect various aspects of the PMR11,12,13,14,15,16,17. For example, based on our current knowledge, the SCs, via the proteins they produce, seem to be required for the perpetuation of the SP signaling after the first day18.
Given the rarity of the SCs (only 80 cells per male), all our knowledge about these cells and the proteins they produce comes from genetics and candidate approaches. Thus far, only a relatively small list of genes has been shown to be SC-specific. This list includes the homeodomain protein Defective proventriculus (Dve)19, the lncRNA MSA20, Rab6, 7, 11 and 1921, CG1656 and CG1757511,15,21 and the homeobox transcription factor Abdominal-B (Abd-B)18. Previously, we have shown that a mutant deficient for both the expression of Abd-B and the lncRNA MSA in secondary cells (iab-6cocuD1 mutant) shortens the length of the PMR from ~10 days to only one day12,18,20. This phenotype seems to be caused by the improper storage of SP in the female reproductive tract12,18,20. At the cellular level, the secondary cells of this mutant show abnormal morphology, losing their characteristic vacuole-like structures18,20,21. Using this mutant line, we previously attempted to identify genes involved in SC function by comparing the transcriptional profiles of whole AGs from either wild type or mutant accessory glands12. Also, other labs showed that SC number, morphology and vacuolar content depend on male diet, mating status and age21,25,26.
Although positive progress was made using these approaches, a full SC transcriptome was far from achieved. The rareness of these cells in this organ made it difficult to progress further even from wild type cells. For testing gene expression, changes in these cells after particular environmental stimuli would be even harder. Thus, a method for isolating and purifying SC RNA that was fast and simple enough to perform under different genetic backgrounds and environmental conditions was needed.
Both the Abd-B and MSA genes require a specific 1.1 kb enhancer from the Drosophila Bithorax Complex (called the D1 enhancer) for their expression in SCs18,20. This enhancer has previously been used to create a GAL4 driver that, when associated with a UAS-GFP, is able to drive strong GFP expression specifically in SCs. Thus, we used this line as the basis for a FACS protocol to isolate these cells from both wild type and iab-6cocuD1 AGs). As iab-6cocuD1 mutant SCs display a different cellular morphology, we show that this protocol can be used to isolate cells for the determination of their transcriptome from this rare cell type under vastly different conditions.

<table><tbody><tr><td>24-wells Tissue Culture plate</td><td>VWR</td><td>734-2325</td><td></td></tr><tr><td>Binocular microscope for dissection</td><td></td><td></td><td></td></tr><tr><td>Binocular with light source for GFP</td><td></td><td></td><td></td></tr><tr><td>Bunsen</td><td></td><td></td><td></td></tr><tr><td>Draq7 0.3 mM</td><td>BioStatus</td><td>DR71000</td><td></td></tr><tr><td>Plastic microtubes 1.5 mL</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>FACS</td><td>Beckman Coulter</td><td>MoFlo Astrios</td><td></td></tr><tr><td>Fine dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Foetal bovine serum</td><td>Gibco</td><td>10270-106</td><td>heat inactivated prior to use</td></tr><tr><td>Glass dishes for dissection</td><td></td><td></td><td></td></tr><tr><td>Ice bucket</td><td></td><td></td><td></td></tr><tr><td>ImProm-II Reverse Transcription System</td><td>Promega</td><td>A3800</td><td></td></tr><tr><td>MasterPure RNA Purification Kit</td><td>Epicentre</td><td>MCR85102</td><td></td></tr><tr><td>Nextera XT kit</td><td>illumina</td><td></td><td>&lt;a href=&quot;https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html&quot;&gt;https://emea.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html&lt;/a&gt;</td></tr><tr><td>P1000</td><td>Gilson</td><td></td><td></td></tr><tr><td>P20</td><td>Gilson</td><td></td><td></td></tr><tr><td>P200</td><td>Gilson</td><td></td><td></td></tr><tr><td>Papain</td><td></td><td></td><td>50U/mL stock</td></tr><tr><td>PBS</td><td></td><td></td><td>home made</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>PolydT primers</td><td></td><td></td><td></td></tr><tr><td>Random hexamer primers</td><td></td><td></td><td></td></tr><tr><td>RNA 6000 Pico kit</td><td>Agilent</td><td></td><td></td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; medium</td><td>Gibco</td><td>21720-001</td><td></td></tr><tr><td>SMARTer cDNA synthesis kit</td><td>Takara</td><td></td><td>&lt;a href=&quot;https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits&quot;&gt;https://www.takarabio.com/products/cdna-synthesis/cdna-synthesis-kits/smarter-cdna-synthesis-kits&lt;/a&gt;</td></tr><tr><td>SYBR select Master mix for CFX</td><td>applied biosystems</td><td>4472942</td><td></td></tr><tr><td>Thermo Shaker</td><td>Hangzhou Allsheng intruments</td><td>MS-100</td><td></td></tr><tr><td>Tipone 1250 &amp;mu;L graduated tip</td><td>Starlab</td><td>S1161-1820</td><td></td></tr><tr><td>Tipone 200 &amp;mu;L bevelled tip</td><td>Starlab</td><td>S1161-1800</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco</td><td>12604013</td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr></tbody></table>

a facs-based protocol to isolate rna from the secondary cells of drosophila male accessory glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

The protocol presented here enables one experimenter to isolate secondary cells from Drosophila male accessory glands and to extract their RNA in the course of one single day (Figure 1).
We use the Abd-B-GAL4 construct18 to label secondary cells (SC) but not main cells (MC) with GFP (Figure 2A). One objective of...

Watch this Scientific Journal Video about A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands at JoVE.com

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

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Research

JoVE Journal

Genetics

6.9K Views.  University of Geneva. Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

Video: A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Laser Capture Microdissection of Drosophila Peripheral Neurons

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.

Laser Capture Microdissection of Drosophila Peripheral Neurons

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.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Neuroscience

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Developmental Biology

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Biology

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

Quantifying the Poly(A) Length of Genes from the Drosophila Nervous System

Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is tightly regulated by cellular processes. Dysregulation of mRNA polyadenylation has been associated with abnormal gene expression and various diseases, including cancer, neurological disorders, and developmental abnormalities. Therefore, comprehending the dynamics of polyadenylation is vital for unraveling the complexities of mRNA processing and posttranscriptional gene regulation. 
This paper presents a method for measuring poly(A) tail lengths in RNA samples isolated from Drosophila larval brains and Drosophila Schneider S2 cells. We employed the guanosine/inosine (G/I) tailing approach, which involves the enzymatic addition of G/I residues at the 3' end of mRNA using yeast poly(A) polymerase. This modification protects the RNA's 3' end from enzymatic degradation. The protected full-length poly(A) tails are then reverse-transcribed using a universal antisense primer. Subsequently, PCR amplification is performed using a gene-specific oligo that targets the gene of interest, along with a universal sequence oligo used for reverse transcription. 
This generates PCR products encompassing the poly(A) tails of the gene of interest. Since polyadenylation is not a uniform modification and results in tails of varying lengths, the PCR products display a range of sizes, leading to a smear pattern on agarose gel. Finally, the PCR products are subjected to high-resolution capillary gel electrophoresis, followed by quantification using the sizes of the poly(A) PCR products and the gene-specific PCR product. This technique offers a straightforward and reliable tool for analyzing poly(A) tail lengths, enabling us to gain deeper insights into the intricate mechanisms governing mRNA regulation.

Author Spotlight: Exploring the Frontier of mRNA Research with Poly A Tail Analysis Techniques

The protocol describes an efficient and reliable method for quantifying the poly(A) length of the gene of interest from the Drosophila nervous system, which can be easily adapted to tissues or cell types from other species.

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

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Rozdziały w tym wideo

Laser Capture Microdissection of Drosophila Peripheral Neurons

Purification of Low-abundant Cells in the Drosophila Visual System

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line