Funding support for this project was provided by a grant to HMK by the Canadian Institutes of Health Research (grant MOP 133473).

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Rev Genet. 10, 57-63 (2009).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98, 81-85 (1989).</li><li>Hughes, S. C., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+labeling+with+fluorescence+in+situ+hybridization+in+Drosophila+whole-mount+embryos.">Double labeling with fluorescence in situ hybridization in Drosophila whole-mount embryos.</a> Biotechniques. 24, 530-532 (1998).</li><li>Lecuyer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Kosman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Raap, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-sensitive+FISH+using+peroxidase-mediated+deposition+of+biotin-+or+fluorochrome+tyramides.">Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.</a> Human Mol Genet. 4, 529-534 (1995).</li><li>Lecuyer, E., Parthasarathy, N., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+protocols+in+Drosophila+embryos+and+tissues.">Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.</a> Methods Mol Biol. 420, 289-302 (2008).</li><li>Wilk, R., Murthy, S. U. M., Yan, H., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols Essential Laboratory Techniques. , 1-9 (2010).</li><li>Holt, C. E., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+mRNA+Localization+in+Animal+Cells+and+Why+It+Matters.">Subcellular mRNA Localization in Animal Cells and Why It Matters.</a> Science. 326, 1212-1216 (2009).</li><li>Martin, K. C., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+Localization:+Gene+Expression+in+the+Spatial+Dimension.">mRNA Localization: Gene Expression in the Spatial Dimension.</a> Cell. , 719-730 (2009).</li><li>Wilk, R., Hu, J., Blotsky, D., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+and+pervasive+subcellular+distributions+for+both+coding+and+long+noncoding+RNAs.">Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.</a> Genes Dev. 30, 594-609 (2016).</li><li>Zhou, X. L., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximo-distal+specialization+of+epithelial+transport+processes+within+the+Xenopus+pronephric+kidney+tubules.">Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules.</a> Dev Biol. 271, 322-338 (2004).</li></ol>

Authors have no competing financial interests.

The described protocol provides a highly reproducible, sensitive, and economical method for the detection of most RNAs in fixed Drosophila embryos or tissues. Although slightly more complicated than the more traditionally used alkaline phosphatase method of probe detection, the resolution obtained by FISH is far greater and the sensitivity is comparable or improved 7,8. By using whole or partial microtiter plates, the protocols can be used for large-scale or limited analyses of genes of interest. It should be noted that the probes generated may detect all or multiple transcript splice forms unless probes are more specifically designed (i.e., single exons). Although we have tested probes as small as 200 nucleotides with reasonable success, smaller probes tend to be less effective and may be less specific. Clearly, this protocol is not appropriate for the detection of small introns, exons, or processed microRNAs.
Although this approach uses an enzymatic step to boost the strength of signals, signal intensity appears to be proportional to target gene expression in a relatively linear and broad range of expression levels. At higher magnification, the signal is seen as puncta or speckles within cells and tissues. For relatively rare transcripts only a few tiny puncta are seen. As transcript abundance increases, these grow in number and size. As with single molecule FISH (smFISH), single small puncta may also correspond to single RNAs and should also be readily quantified using imaging and informatics software. Comparisons of published images obtained using smFISH with images that we have curated for the same targets suggest that the overall, sensitivity and resolution of the two methods are relatively similar, although this should be tested by more rigorous comparisons. Given the much higher expense of smFISH, the method provided here should give similar results at a fraction of the cost. However, if the goal is to detect small transcripts, or distinct parts of transcripts, smFISH is recommended.
Due to the smaller size of some FISH probes, this method may also be better at penetrating tissues that are large or have significant barriers. However, our use of higher detergent levels and tissue fixation and permeabilization tweaks appear to have solved this problem. Finally, although developed for Drosophila tissues, the high detergent buffers described here for dissected tissues should also work for Drosophila embryos as well as most other organisms and tissues.

New genome-wide RNA detection methods such as RNA-seq have greatly expanded our knowledge of when and where genes are expressed, and at what levels1. However, these provide relatively poor temporal and spatial resolution. RNA in situ hybridization allows the spatial distribution of transcripts to be visually observed in fixed tissues, thus revealing the details of cellular and subcellular localization2. Using fluorescent in situ hybridization (FISH) allows even more spatial resolution as it allows the use of powerful microscopy techniques, such as confocal and light sheet microscopy3. Fluorescent markers such as DAPI can also be used to establish subcellular relationships4. FISH also facilitates the observation of multiple RNAs and proteins simultaneously with clear indications of overlap at the subcellular level. Unique reagents and steps required for double-labeling can be found in the following references3,5.
The procedures described here utilize 96-well microtiter plates for all steps, including cDNA template production (colony PCR), RNA probe production (using T7, T3 or SP6 polymerase-dependent run-off transcription), in situ hybridization, and fluorescent signal development. Sufficient numbers of embryos or tissues are added to each well of the 96-well plate to allow evaluation of consistency and variability. Each well then receives a different probe. After signal development, embryos or tissues from each well are arrayed on individual microscope slides for microscopic analysis.
The use of tyramide signal amplification (TSA) for probe detection produces robust signals with exceptional subcellular resolution 5,6,7,8. The subcellular localization of RNAs is an important regulatory mechanism 9 that appears to occur with virtually all RNAs 4,10,11. These analyses have also shown that many transcripts that are not detected by RNA-seq are readily detected by FISH 8. The adaptation of RNA in situ hybridization to 96-well plates allows analysis of up to 96 genes of interest at a time (more if additional plates are used), making analysis of large datasets possible. By cutting the plates into smaller sections, the method is also easily adapted to just a few samples. The protocol provided is appropriate for analysis of RNA distributions in most types of tissues. Although not shown, it is adaptable to non-Drosophila tissues as well.

<table>
	<tbody>
		<tr>
			<td>15 ml Polypropylene conical centrifuge tubes</td>
			<td>Frogga Bio</td>
			<td>TB15-500</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>2x PCR Master Mix with dye(Ambiogene)</td>
			<td>Biomart (Canada)</td>
			<td>141106-1K</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical centrifuge tubes</td>
			<td>Frogga Bio</td>
			<td>TB50-500</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>96-well filteration plate (1.2 um, hydrophilic PVDF membrane</td>
			<td>EMD MILLIPORE</td>
			<td>MSBVS1210</td>
			<td>Used to remove liguid from bottom of the plate with floting tissues</td>
		</tr>
		<tr>
			<td>96-well PCR plate (200 &#956;l)</td>
			<td>Denville</td>
			<td>C18096-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc syringe filter (0.2 u)</td>
			<td>PALL</td>
			<td>PN4612</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc syringe filter (0.45 u)</td>
			<td>PALL</td>
			<td>PN4614</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA002.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Sigma-aldrich</td>
			<td>A9393-5G</td>
			<td>Stock concentration is 100 g/ml disolved ddH&#8322;O and 0.2&#956;m filtered (use as 1/1000)</td>
		</tr>
		<tr>
			<td>AXYGEN 96-well assay storage plates</td>
			<td>UltiDent Scientific</td>
			<td>24-P96-450V-C</td>
			<td>To store bacterial glycerol stocks</td>
		</tr>
		<tr>
			<td>AXYGEN Aluminum Plate Seals</td>
			<td>UltiDent Scientific</td>
			<td>24-PCR-AS-200</td>
			<td>To seal plates</td>
		</tr>
		<tr>
			<td>AXYGEN Wide-bore tips (200 ul)</td>
			<td>UltiDent Scientific</td>
			<td>24-T205-WB-C</td>
			<td>To transfer samples</td>
		</tr>
		<tr>
			<td>Biotin-SP (long spacer) IgG Fraction Monoclonal Mouse Anti-Digoxin</td>
			<td>Jackson ImmunoResearch Lab</td>
			<td>200062156</td>
			<td>Primary antibody for in situ (stock conc. 1 mg/ml, working conc. 2.5 &#956;g/ml or 1/400 dilution.</td>
		</tr>
		<tr>
			<td>BRAND 8-channel manifold (autoclavable)</td>
			<td>BrandTech Scientific Inc.</td>
			<td>704526</td>
			<td>To remove liquids from the top of 96-well PCR plates</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-aldrich</td>
			<td>C1919</td>
			<td>Stock concentration 34 mg/ml dissolved and 0.2&#956;m filtered in 100 % EtOH (use as 1/1000)</td>
		</tr>
		<tr>
			<td>Cyanine 3 Tyramide (Cy3-TSA)</td>
			<td>Home made</td>
			<td></td>
			<td>Could be ordered from Perkin-Elmer Cat# SAT704A001EA</td>
		</tr>
		<tr>
			<td>DABCO (1,4-Diazabicyclo[2.2.2]octane)</td>
			<td>Sigma-aldrich</td>
			<td>D2522</td>
			<td>Antifade reagent in mounting media</td>
		</tr>
		<tr>
			<td>DEPC (Diethyl pyrocarbonate)</td>
			<td>Sigma-aldrich</td>
			<td>D5758</td>
			<td>Working concentration is 0.1 % in ddH&#8322;O. To make RNase free water 0.1 %</td>
		</tr>
		<tr>
			<td>Digoxigenin-11-uridine-5&#8217;-triphosphate</td>
			<td>Roche</td>
			<td>11-209-256-910</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo collection cage</td>
			<td>Home made</td>
			<td>N/A</td>
			<td>Size: 75 cm X 75 cm X 75 cm made with Polymethyl methacrylate</td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5804</td>
			<td>Eppendorf</td>
			<td>Model 5804</td>
			<td>Centrifuge for 96-well plates on A-Z-DWP rotor</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Bioshop</td>
			<td>FOR001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bioshop</td>
			<td>GLY002.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bioshop</td>
			<td>GLN001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H4784-250MG</td>
			<td>Stock: 50 mg/ml. Working concentration 1 mg/ml in Hybridization solution</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H&#8322;O&#8322;)</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Armor Beads</td>
			<td>DiaMed</td>
			<td>LAA42370-001</td>
			<td>Fill in heating unit</td>
		</tr>
		<tr>
			<td>Labnet Gyro M Mini Nutating Mixer</td>
			<td>Fisher Scientific</td>
			<td>50-998-347</td>
			<td>Sample mixing</td>
		</tr>
		<tr>
			<td>Mesh - 100 micons</td>
			<td>FlyStuff</td>
			<td>57-103</td>
			<td>For collecting embryos</td>
		</tr>
		<tr>
			<td>Mesh - 800 microns</td>
			<td>SEFAR - Nytex</td>
			<td>06-780/53</td>
			<td>For catching loose tissue / may be used for special tube in figure 4.</td>
		</tr>
		<tr>
			<td>Micro Cover Glasses</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td>Size: 22 X 22 mm</td>
		</tr>
		<tr>
			<td>Micro slides,frstd</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Size: 25 X 75 mm</td>
		</tr>
		<tr>
			<td>Multi-Well Plate Vacuum Manifold</td>
			<td>Pall Corporation</td>
			<td>P/N 5017</td>
			<td>Remove liquid from bottom of 96-well filter plate</td>
		</tr>
		<tr>
			<td>Paraformaldehyd (PFA)</td>
			<td>Bioshop</td>
			<td>PAR070.500</td>
			<td>Fixation</td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Bio-Rad</td>
			<td>Model: DNA Engine PTC-200</td>
			<td>96-well plate PCR and probe denature</td>
		</tr>
		<tr>
			<td>Picric acid solution</td>
			<td>Sigma-aldrich</td>
			<td>80456</td>
			<td>For tissue fixation I solution</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCL)</td>
			<td>Bioshop</td>
			<td>POC308</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH&#8322;PO&#8324;)</td>
			<td>Bioshop</td>
			<td>PPM302.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PP lid for 96-well plates, 100/case</td>
			<td>UltiDent Scientific</td>
			<td>24-P-LID-PP</td>
			<td>Lid for storing plates</td>
		</tr>
		<tr>
			<td>Progene disposable Reagent Reservoirs (50 ml, sterile)</td>
			<td>UltiDent Scientific</td>
			<td>825-1-50-5S</td>
			<td>Liquid handling</td>
		</tr>
		<tr>
			<td>Progene Pierceable Aluminum Foil</td>
			<td>UltiDent Scientific</td>
			<td>87-CFILM-AL</td>
			<td>Seal 96-well plate, DNase and RNase free</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-aldrich</td>
			<td>P2308- 5MG</td>
			<td>Embryo permeabilization</td>
		</tr>
		<tr>
			<td>RiboLock Ribonuclease Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td>40 Unit/ul</td>
		</tr>
		<tr>
			<td>Ribonucleoside Triphosphate (NTP) set</td>
			<td>Roche</td>
			<td>11277057001</td>
			<td>RNA probe sythesis</td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>GIBCO</td>
			<td>10977015-500ml</td>
			<td>RNA probe sythesis</td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Ambion</td>
			<td>AM9780</td>
			<td>Remove RNase contamination</td>
		</tr>
		<tr>
			<td>Single stranded DNA from salmon testes</td>
			<td>Sigma-aldrich</td>
			<td>D9156</td>
			<td>For hybridization solution</td>
		</tr>
		<tr>
			<td>Skim milk (non fat power)</td>
			<td>Bioshop</td>
			<td>SKI400.500</td>
			<td>Working concentration is 1 % in 1 X PBT or PBTT as a blocking reagent</td>
		</tr>
		<tr>
			<td>Sodium Acetate (NaOAc) 3 M, pH=5.2 (RNase free)</td>
			<td>Bioshop</td>
			<td>SAA333</td>
			<td>Working concentration is 10 % (v/v) for DNA or RNA precipitation</td>
		</tr>
		<tr>
			<td>Sodium citrate (HOC(COONa)(CH&#8322;COONa)&#8322; &#183; 2H&#8322;O</td>
			<td>Sigma-aldrich</td>
			<td>S4641-500G</td>
			<td>Reagent in 20 X SSC</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na&#8322;HPO&#8324;)</td>
			<td>Bioshop</td>
			<td>SPD307.1</td>
			<td>Reagent in 10 X PBS</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>EP0131</td>
			<td>20 Unit/ul</td>
		</tr>
		<tr>
			<td>Stainless Steel Shot for Sand Bath</td>
			<td>VWR</td>
			<td>13259-274.</td>
			<td>To fill the heating unit</td>
		</tr>
		<tr>
			<td>Stereomicroscope &#38; gooseneck light source</td>
			<td>Leica</td>
			<td>Model: MZ6</td>
			<td>Tissue dissection and mounting</td>
		</tr>
		<tr>
			<td>Streptavidin, horseradish peroxidase (HRP) conjugate</td>
			<td>Molecular Probes</td>
			<td>S911</td>
			<td>Secondary antibody stock: 1 mg/ml, use 1/1000 dilution (1 &#956;g/ml) for in situ.</td>
		</tr>
		<tr>
			<td>T3 RNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>EP0101</td>
			<td>20 Unit/ul</td>
		</tr>
		<tr>
			<td>T7 RNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>EP0111</td>
			<td>20 Unit/ul</td>
		</tr>
		<tr>
			<td>Tip station (10 ul)</td>
			<td>Axygen</td>
			<td>T-300-STK-S</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>Tip station (200 ul)</td>
			<td>Sorbio</td>
			<td>27770T</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>Tips ( 1ml)</td>
			<td>Sorbio</td>
			<td>10200</td>
			<td>Certified DNase and RNase free</td>
		</tr>
		<tr>
			<td>TRITON X-100</td>
			<td>Bioshop</td>
			<td>TRX506</td>
			<td>Working concentration is 0.3% in 1 X PBT for tissue in situ</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bioshop</td>
			<td>TWN510</td>
			<td>Working concentration is 0.1% in 1 X PBT for embryo in situ</td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper, Grade 3</td>
			<td>Sigma-aldrich</td>
			<td>WHA1003917</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td>Preparation</td>
		</tr>
		<tr>
			<td>DABCO (1,4-Diazabicyclo[2.2.2]octane) glycerol anti-fade mounting media</td>
			<td></td>
			<td></td>
			<td>DABCO (1.25 g), Glycerol (35 ml or v/v 70 %) and 1 X PBS (15 ml, or v/v 30 %). Keep dark and rock in cold room. store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>DEPC (Diethyl pyrocarbonate) treated ddH&#8322;O (1L)</td>
			<td></td>
			<td></td>
			<td>Add 1 ml of DEPC in 999 ml of ddH&#8322;O (0.1 %). Stir O/N in fumehood O/N and then autoclave 45 min liquid cycle</td>
		</tr>
		<tr>
			<td>10 X PBS (1L)</td>
			<td></td>
			<td></td>
			<td>NaCL (80 g), KCL (2 g), Na&#8322;HPO&#8324; (14.4 g), KH&#8322;PO&#8324; (2.4 g) and unautoclaved DECP ddH&#8322;O (800 ml). Adjust pH to 7.4 with 10 N NaOH and then bring the total volume to 1 L. Autoclave 45 min liquid cycle</td>
		</tr>
		<tr>
			<td>20 X SSC (1L)</td>
			<td></td>
			<td></td>
			<td>NaCL (175.3 g), Sodium citrate tribasic dihydrate(88.2 g) and unautoclaved DECP ddH&#8322;O (800 ml). Adjust pH to 7.4 with 10 N NaOH and then bring the total volume to 1 L. Autoclave 45 min liquid cycle</td>
		</tr>
		<tr>
			<td>1 X PBT (1L)</td>
			<td></td>
			<td></td>
			<td>1 X PBS with 1 ml Tween 20 (0.1 %)</td>
		</tr>
		<tr>
			<td>1 X PBTT (1L)</td>
			<td></td>
			<td></td>
			<td>1 X PBS with 1 ml Tween 20 (0.1 %) and 3 ml of Triton-X-100 (0.3 %)</td>
		</tr>
		<tr>
			<td>1 X PBTB (1L)</td>
			<td></td>
			<td></td>
			<td>1 % Skim milk in 1 X PBT</td>
		</tr>
		<tr>
			<td>1 X PBTTB (1L)</td>
			<td></td>
			<td></td>
			<td>1 % Skim milk in 1 X PBTT</td>
		</tr>
	</tbody>
</table>

high resolution fluorescent in situ hybridization in drosophila embryos and tissues using tyramide signal amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

Figure 6 shows examples of results obtained using this procedure. The top 3 panels show examples of early Drosophila embryos (Figure 6, panels A-C), the next 3 panels show examples of late Drosophila embryos with signals in different tissues (amnioserosa, muscles and central nervous system, respectively (Figure 6, panels D-F). Next are examples from 3rd instar larval tissues (Figure 6, panels G-J). Panels K and L show representative images from adult gonads (ovary and testis, respectively). Hundreds of images have been uploaded and annotated in our searchable &#39;fluorescent in situ fruit fly database&#39; (FlyFISH (http://fly-fish.ccbr.utoronto.ca)).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56281/56281fig1.jpg" /> 
Figure 1. Outline of Fluorescent in situ hybridization (FISH) protocol and key equipment. (A) PCR amplification of cDNA. (B) In vitro transcription to generate gene-specific antisense DIG- labeled RNA probes in a 96-well plate (photo of the plate shown in b). (C) and (D): Adults (female: male ratio 2:1. 300-400 flies/bottle) flies are allowed to mate for 3-4 days. Embryos from an overnight collection on standard cornmeal food at 25 &#176;C are transferred to a well ventilated plastic box (1 L of cornmeal food in a container sized: 8&#34; X 8&#34; X 3&#34; LWH) or to new bottles keeping a proper larval density. For tissue collection, active yeast powder should be sprinkled on top of the food at 24, 48 h after egg laying (AEL). (E to H): Fluorescent in situ hybridization experiments performed over 3 consecutive days. For embryos or tissues that do not float, use a 96-well PCR plate as shown in photograph b with 8-channel aspirator (photograph c). For tissues that float, like most larval tissues, use a filter-bottomed plate (photograph in d) and remove liquid from bottom with a manifold aspirator using low pressure (photograph in e). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56281/56281fig2.jpg" /> 
Figure 2. Determination of RNA probe yield. Newly synthesized antisense RNA probes observed on a 1 % agarose gel (5 &#181;L/lane). The yield can be categorized (based on the intensity of the band) as &#39;low yield&#39; in lanes 3 and 10, &#39;typical yield&#39; in lanes 1, 4, 5, 6 and 8, or &#39;high yield&#39; in lanes 2, 7, 9, 11, and 12. For samples with &#39;typical yields&#39;, use 10-15 &#181;L of probe diluted in 100 &#181;L of hybridization solution for each in situ experiment. Dilute samples with &#39;high yields&#39; of probe with additional hybridization solution according to the band intensity relative to a &#39;typical probe&#39; intensity. Aim to have roughly equal final probe concentrations in each well. The arrow points to one of the &#39;RNA probes&#39;, the higher band on each lane is the original DNA template. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56281/56281fig3.jpg" /> 
Figure 3. Major steps for a large-scale embryo collection and fixation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56281/56281fig4.jpg" /> 
Figure 4. Homemade tube for removing liquid from larval or floating tissues. Some larval and adult tissues float in solution during fixation. This simple tool consists of a nylon mesh held by a 200 &#181;L cut tip, followed by a 1 mL tip as an adaptor to connect to an aspirator. Liquid can be safely removed without losing any tissue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56281/56281fig5.jpg" /> 
Figure 5. Tyramide Signal Amplification. (1) DIG-labeled antisense RNA probe hybridizes with endogenous sense RNA. (2) Immuno-affinity binding between biotin-conjugated primary antibody and DIG-UTP. (3) HRP conjugated streptavidin binds biotin. (4) HRP catalyzes the cyanine 3-tyramide and hydrogen peroxide reaction to form short-lived cyanine 3-tyramide radicals. (5) Cyanine 3-tyramide radicals bind to tyrosine residues on nearby proteins. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56281/56281fig6.jpg" /> 
Figure 6. Representative in situ hybridization images. Each panel shows an example of a different RNA (shown in Cyan) and nuclei stained with DAPI (shown in red). (A-C) Examples of early Drosophila embryos. (D, F) Examples of late Drosophila embryos. (G-J) Examples of 3rd instar Drosophila larval tissues (malphigian tubules, midgut, salivary gland and muscle respectively). (K) Adult ovary. (L) Adult testis. Each panel displays the name of the gene that the probe is hybridizing to. Scale bars = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56281/56281fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification at JoVE.com

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

high-resolution-fluorescent-situ-hybridization-drosophila-embryos

Nanyang Technological University

Research

JoVE Journal

Genetics

12.8K Views.  University of Toronto. The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

Video: High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...