We would like to thank Erika Taylor for editing this manuscript. This work was funded by grants from the National Institutes of Health (P01 HL098053, P01 AG033561 and R01 HL054732) to R.B., a post-doctoral research supplement (R01 HL085481) and fellowship (AAUW) to S.B.D., and grants from the American Heart Association to S.B.D. and R.T.B.

1. HFD Feeding Protocol
<img alt="Table 1" src="/files/ftp_upload/56029/56029table1.jpg" /> 
Table 1. Fly food recipe. 
This table summarizes the different ingredients used to prepare our control food. Once made, 10 mL of the food is poured into vials, cooled and stored at 4 &#176;C for long term storage.
<ol>
	<li>HFD preparation
	<ol>
		<li>To make 1 kg of high fat food, weigh 700 g of pre-made normal fly food (see Table 1) and 300 g of coconut oil into two separate containers using an analytical balance.</li>
		<li>Heat each container individually in the microwave, until the contents melt completely. Pour the coconut oil into the fly food container. Stir using a whisk until the oil is well mixed into the fly food. 
		NOTE: No chunks of food or oil should be visible. When the fly food is melting in the microwave, stop at intervals of 1 min to mix the food and prevent boiling over.</li>
		<li>Re-weigh the high fat food. If the total weight is less than 1 kg (700 g of food + 300 g of coconut oil), add water (around 10 mL) to compensate for evaporation during heating and to bring back the weight to 1 kg.</li>
		<li>Pour about 10 mL well-mixed high fat food per vial (around 25% of vial volume). Next, cover with cheesecloth to prevent flies&#39; contamination. Cool for 1 h at room temperature then transfer the vials to 4 &#176;C for storage up to 4 weeks. 
		NOTE: The Normal food (NF) is used as a control food. Also pour 10 mL (around 25% of vial volume) of NF per vial. For the HFD food, replace 30% of the NF food with coconut oil. For control and experimental conditions, the flies were given the same amount of NF and HFD (10 mL of food per vial).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56029/56029fig1.jpg" /> 
Figure 1. HFD feeding in Drosophila. 
The scheme shows the different feeding steps for flies on a control food (normal diet without addition of coconut oil-NF) or HFD (with the addition of coconut oil). The entire process takes 10 days after the initial collection of adult flies. <a href="//cloudfront.jove.com/files/ftp_upload/56029/56029fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>HFD feeding of D. melanogaster 
	NOTE: This protocol includes control food and HFD feeding procedures that are compatible with any fly line. Control and experimental flies are put in vials containing the same amount food (NF or HFD).
	<ol>
		<li>Use CO2 to anesthetize the flies. 
		NOTE: For neophytes, please read reference21 for an overview on raising and handling D. melanogaster.</li>
		<li>Collect 0-5 day old flies from vials and transfer (by flipping) the flies into new vials containing NF (previously warmed at room temperature). Let the flies age for 5 additional days at 21 &#176;C.</li>
		<li>At the end of the 5 days, take out the vials containing HFD and NF from 4 &#176;C. 
		NOTE: As cold temperatures can stress living flies, let the vials sit for 30 min at room temperature to warm up before use.</li>
		<li>Using wipes, clean and remove excess fluid from the sides of the vials.</li>
		<li>Insert a small piece of filter paper, about 1 cm x 3 cm, into the high fat food to absorb any surplus liquid. 
		NOTE: This step is critical for preventing the flies from sticking to the HFD food.</li>
		<li>Next, transfer the flies onto the high fat food (by flipping). Lay the vials horizontally on their side, (not standing) at 21-22 &#176;C for 5 days. 
		NOTE: Avoid higher temperatures as this can partially melt the high fat food, causing the flies to stick to the food and die.</li>
		<li>After 5 days, transfer (by flipping) the flies from NF and HFD into new vials without food to allow their guts to empty for 30 min. Next, proceed to weighing the flies. 
		NOTE: One of the critical steps in this HFD feeding protocol is to prepare a well-mixed HFD, free of any chunks of fly food or condensed oil. Homogenous mixing and cooling the HFD to 45-30 &#176;C is advisable, before pouring into vials and storing the food at 4 &#176;C. Proper age matching of control and experimental flies is important as well as controlled nutritional and environmental conditions prior to the HFD feeding. During the feeding phase, control and experimental flies must be given the same amount of NF and HFD. Never collect flies from overcrowded vials or bottles (to avoid transgenerational effects). Always put a maximum of 25 flies per vial during NF and HFD feeding to avoid dietary restriction or other crowding effects. The HFD is composed of 30% coconut oil, thus temperatures above 22 &#176;C might partially melt the HFD, causing the flies to stick on the oily food and die. Before putting the flies on the HFD, clean the vials to remove any residual oil and insert a filter paper to absorb any excess fluid in the food. During the incubation of the flies on HFD, the vials are laid on their sides to provide a fat-free horizontal surface for the flies.</li>
	</ol>
	</li>
	<li>Weighing the flies
	<ol>
		<li>For each genotype and gender to be tested, use an ultra-sensitive scale to record the weights of pre-labeled, empty 1.7 mL tubes.</li>
		<li>Using fly anesthetic or CO2, anesthetize and collect 36 flies of the same genotype, gender, and feeding status with a brush into one pre-weighed 1.7 mL tube.</li>
		<li>Weigh the tube containing flies with the same ultra-sensitive scale.</li>
		<li>Determine the average fly weight following this formula: 
		<img alt="Equation 1" src="/files/ftp_upload/56029/56029eq1.jpg" /> 
		NOTE: The number of flies in this protocol is 36.</li>
		<li>Flash freeze the 1.7 mL tubes containing flies by plunging into liquid nitrogen for 2 min. Collect and put the tubes into boxes. Next, transfer the boxes to -80 &#176;C (preferable temperature) for storage until use with the TAG assay. 
		CAUTION: Liquid nitrogen is at an extremely low temperature. Please use appropriate personal protective gear.</li>
	</ol>
	</li>
</ol>
2. TAG Assay
<ol>
	<li>Preparation of the TAG standard concentrations
	<ol>
		<li>Prepare 500 mL of PBT (PBS 1X, 0.05% Triton).</li>
		<li>Using 0.5 mL tubes and the TAG solution (Table of Materials), prepare 100 &#181;L of blank (PBT only) and 100 &#181;L of six standard TAG concentrations (2 &#181;g/&#181;L; 1 &#181;g/&#181;L; 0.5 &#181;g/&#181;L; 0.25 &#181;g/&#181;L; 0.125 &#181;g/&#181;L; 0.0625 &#181;g/&#181;L) with the PBT.</li>
		<li>Place the tubes on ice and proceed with grinding the flies.</li>
	</ol>
	</li>
	<li>Grinding the flies
	<ol>
		<li>Take out the frozen flies from -80 &#176;C (step 1.3). Transfer the tubes containing the flies on ice.</li>
		<li>First, place 2 mm metal grinding balls into a 96-well ball dispenser, use a small paintbrush to remove extra balls.</li>
		<li>Place the 96-well grinding plate upside down on top of the ball dispenser and flip to transfer the metal balls directly into the grinding plate. There should be one ball per well.</li>
		<li>Using a multi-channel pipette, add 600 &#181;L of PBT into each row of the 96-well grinding plate.</li>
		<li>With forceps, add 3 flies per well. Indicate the genotype, gender and food condition of the flies in each row/well of the 96-well grinding plate.</li>
		<li>Tightly place the caps onto the 96-well grinding plate. Tape may be placed on top to ensure that there is no leakage.</li>
		<li>Properly place the 96-well grinding plates into the grinding machine. Set the machine to the highest speed and press &#34;run&#34; to grind the flies for 3 min.</li>
		<li>After 3 min, stop the grinding and proceed with plate centrifugation: centrifuge for 15 min at 4,000 x g, 4 &#176;C. After centrifugation, manage the grinding plate carefully to avoid mixing the supernatant with the pellet.</li>
	</ol>
	</li>
	<li>Sample loading and TAG content determination
	<ol>
		<li>Take a new 96-well spectrophotometer plate. Using a multi-channel pipette, load 200 &#181;L of the TAG reagent into each row of the plate.</li>
		<li>Load 20 &#181;L of PBT into the first well of the first row, to create a blank. Mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Load 20 &#181;L of each standard dilution (from step 2.1.2) into the remaining wells of the first row and mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Using a multi-channel pipette, transfer 20 &#181;L of supernatant from each row of the grinding plate to the corresponding row in the 96-well spectrophotometer plate. Pipette up and down to mix well. Avoid forming bubbles.</li>
		<li>Next, place a gas-permeable foil over the top of the 96-well plate.</li>
		<li>Incubate the plate at 37 &#176;C for 10 min. 
		NOTE: If there are bubbles in the wells, centrifuge the plate for 2 min at 2,000 x g to remove all bubbles. The presence of bubbles will interfere with the absorbance reading.</li>
		<li>Read the absorbance of each well of the plate at 550 nm using a compatible microplate reader. Next, trace the standard curve and determine the concentration [&#181;g/&#181;L] of the unknown samples.</li>
		<li>To determine the amount of TAG content per mean fly weight, use the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/56029/56029eq2.jpg" /> 
		NOTE: In this protocol, 3 flies were minced in 600 &#181;L of PBT; in consequence, the mean volume per fly is 200 &#181;L.</li>
	</ol>
	</li>
	<li>Bradford assay and normalization of TAG content with protein content
	<ol>
		<li>Prepare seven 100 &#181;L of bovine serum albumin (BSA) standard dilutions (75 &#181;g/mL; 100 &#181;g/mL; 150 &#181;g/mL; 200 &#181;g/mL; 250 &#181;g/mL; 500 &#181;g/mL; 1,000 &#181;g/mL) with the PBT.</li>
		<li>Take a new 96-well plate and load 200 &#181;L of 1X Bradford reagent into each row of the plate.</li>
		<li>In the first well of the first row, add 10 &#181;L of PBT to create a blank. Mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Add 10 &#181;L of each standard dilution into the remaining wells of the first row and mix by pipetting up and down. Avoid forming bubbles.</li>
		<li>Using a multi-channel pipette, transfer 10 &#181;L of the fly supernatant from each row of the grinding plate into the corresponding row in the 96-well plate. Pipette up and down to mix well. Avoid forming bubbles.</li>
		<li>Place a gas-permeable foil over the top of the 96-well plate.</li>
		<li>Incubate the plate at room temperature for 5 min. If there are bubbles in the wells, centrifuge the plate for 2 min at 2,000 x g to remove them. 
		NOTE: The presence of bubbles will interfere with the absorbance reading.</li>
		<li>Use a spectrophotometer to read the absorbance of the blank, standards and unknown samples at 595 nm. Use the standard curve to determine the concentrations [&#181;g/&#181;L] of the samples in each row.</li>
		<li>Normalize the TAG content with protein level using the following formula: 
		<img alt="Equation 3" src="/files/ftp_upload/56029/56029eq3.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Obesity induction in mice can take months19,20. In flies, this HFD feeding protocol allows for induction of excess fat accumulation in a matter of days or less, causing increases in fat accumulation only after 18 h (see Figure 2). HFD feeding with the described protocol increases glucose content 12 and decreases Bmm lipase and PGC-1 expression24. This is in contrast to fasting ...

We are in a time where obesity, and its associated economic burdens, is a worldwide problem1. Two out of every three Americans are overweight or obese with related heart pathologies, the primary cause of death within the adult population2. New efficient methods are needed to adequately investigate the genetic and molecular components implicated in the regulation of metabolic syndrome using model organisms. For this reason, we choose the fruit fly Drosophila model because it shares the most basic biological processes with mammals, including mice and humans3,4,5,6. Drosophila&#39;s genome is highly conserved during evolution but overall much smaller with less gene duplication and metabolic complexity, making it ideal for understanding the fundamental mechanisms implicated in many human diseases4,7,8. Also, characteristic processes carried out by adipose tissue, the gut and pancreas are represented in the fly and mediate regulatory functions in glucose and lipid metabolism, for example, that are similar to humans9,10,11. Moreover, the basic molecular pathways involved in the control of obesity, insulin resistance and diabetes in humans are functionally conserved in Drosophila melanogaster12,13,14,15,16. Like higher organisms, Drosophila has a beating heart that is formed during development by similar processes to that of the mammalian heart3,17. Thus, the development of a reliable HFD feeding protocol and high throughput TAG assay, adapted for efficient screening purposes using the genetic tool box of Drosophila, provide an important means to study and understand the fundamental genetic basis underlying complex metabolic diseases.
The HFD food itself is made from a standard laboratory fly food supplemented with coconut oil, which is composed mostly of saturated fatty acids known to be associated with metabolic syndrome18. While inducing obesity in mammalian models, such as rodents, can take months19,20, our optimized HFD feeding protocol in Drosophila effectively and reproducibly increases organismal fat content in a matter of days12,14. This protocol, in conjunction with a high throughput TAG assay, allows efficient mass screening for the effects of genetic factors, environmental influences and drug candidates to discover new modulators of fat metabolism. In consequence, these protocols are likely relevant to understand and/or combat obesity and obesity-associated human pathologies.
The feeding protocol is versatile and may be applied to study the metabolic and functional effects of single saturated or unsaturated fatty acid. The use of this high throughput TAG assay is not limited to D. melanogaster, but may be adapted to a variety of small model organisms with cuticle or tough extracellular matrices (e.g., other Drosophila species, C. elegans and other emerging invertebrate model organisms) to measure fat content under different environmental, genetic or physiological conditions, at any stage of development, adulthood or phase of metabolic disease. The TAG assay is based on a colorimetric measurement of a series of enzymatic reactions that degrade the TAGs into free fatty acids, glycerol, Glycerol 3-phosphate and finally H2O2 that reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2-hydroxybenzene sulfonate (3,5 DHBS) to produce a red colored product that is measured using a 96-well spectrophotometer.

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			<td height="21" style="width:400px;height:21px;">Triglyceride Standard</td>
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			<td height="21" style="width:400px;height:21px;">Quick Start Bradford Protein Assay</td>
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			<td height="21" style="width:400px;height:21px;">Drosophila vials</td>
			<td style="width:209px;">Genesee Scientific</td>
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			<td>1-Ply, 4.4 x 8.4&#34;</td>
		</tr>
	</tbody>
</table>

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.

In D. melanogaster, as is the case with other species, there is sexual dimorphism between males and females22. It is well known that females are larger, with more fat in their abdomens, than males22. To test the effectiveness of our protocol, we performed TAG assays to determine the differences in TAG content between males and females of standard laboratory wildtype (w1118) flies. The data show that females have ...

Watch this Scientific Journal Video about High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster at JoVE.com

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.

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 ...

high-fat-diet-feeding-high-throughput-triacylglyceride-assay

Research

JoVE Journal

Genetics

11.1K Views.  Sanford Burnham Prebys Medical Discovery Institute. 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.

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

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.

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.

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 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...