Part 1: Extraction of Total RNA using TRIzol Reagent
When working with RNA it is very important to work in an environment that is free of RNAses. Simple precautions such as having reserved pipettes for use only with RNA procedures and spraying the work area with a decontaminant reagent (e.g., RNAse Away) before beginning the procedure are very helpful.
<ol>
<li>To attain a sufficient amount of RNA pool 50 zebrafish embryos in a 1.5 ml microfuge tube and remove as much water as possible with a pipette.</li>
<li>Immediately add 250 &mu;l of TRIzol reagent1 to the microfuge tube containing the embryos. TRIzol reagent contains phenol and should only be used under a fume hood.</li>
<li>Lyse and homogenize the embryos with a pellet pestle (approximately 20 strokes) until the tissue is sufficiently disrupted.</li>
<li>When the cells are sufficiently homogenized, add 750 &mu;l TRIzol reagent to equal a total volume of 1 ml.</li>
<li>To permit complete dissociation of nucleoprotein complexes, incubate homogenized samples for 5 minutes at room temperature. At this stage, the sample can be flash frozen in liquid nitrogen and stored at -80&deg;C or the protocol can be continued.</li>
<li>To continue add 0.2 ml of chloroform and rock the tube for 15 seconds to mix.</li>
<li>Incubate the sample for 2 minutes at room temperature and then centrifuge at 12,000 x g for 15 minutes at 4&deg;C.</li>
<li>The mixture will separate into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. The upper aqueous phase contains the RNA. The top layer should comprise of about 60% of the initial volume of TRIzol (approximately 0.6 ml). Transfer the top aqueous layer into a new microfuge tube using a 1 ml pipette being careful not to transfer any of the interphase layer.</li>
<li>To precipitate the RNA add 0.5 ml isopropanol.</li>
<li>Allow the sample to sit at room temperature for 10 minutes and then centrifuge the sample at 12,000 x g for 10 minutes at 4&deg;C. RNA will form a gel-like pellet on the bottom of the tube.</li>
<li>Without disturbing the pellet, remove the supernatant with a pipette and wash the pellet in 1 ml 75% ethanol. Mix the sample by gentle inversion and centrifuge at 7,500 x g for 5 minutes at 4&deg;C.</li>
<li>After centrifugation, remove the ethanol with a pipette and allow sample to dry while inverted for 10 minutes.</li>
<li>Resuspend the pellet by adding 100 &mu;l of RNAse-free water and incubate the sample at 55&deg;C for 10 minutes. It is recommended to finger vortex frequently during incubation to aid in RNA rehydration. Optionally, you can check sample quality and quantity using a NanoDrop ND-1000 spectrophotometer or directly continue to the next step.</li>
</ol>
Part 2: RNA Cleanup using the Qiagen RNEasy Mini Kit
The Qiagen RNEasy Mini Kit2 is useful in removing contaminants and impurities from an RNA sample isolated from other methods. For this procedure, &beta;-mercaptoethanol, ethanol, and nuclease-free water are needed in addition to the components included in the kit. An optional DNAse treatment is recommended during this procedure.
<ol>
<li>Upon first use of the kit, Buffer RPE will need to be prepared by adding 4 volumes of 100% ethanol to 1 volume of Buffer RPE.</li>
<li>On the day of the procedure, Buffer RLT will need to be prepared fresh. Calculate the total amount of Buffer RLT that will be needed. A volume of 350 &mu;l is needed per sample. To prepare Buffer RLT, add 10 &mu;l of &beta;-mercaptoethanol for every 1 ml of buffer. This should be done under the fume hood.</li>
<li>To each sample add 350 &mu;l of Buffer RLT and 250 &mu;l of 100% ethanol. Mix well by pipetting up and down several times.</li>
<li>Transfer the sample, which should be approximately 700 &mu;l, into an RNEasy column that is placed in a 2 ml collection tube and centrifuge at 8,000 x g for 1 minute at room temperature.</li>
<li>After centrifuging, discard the flow through and add 700 &mu;l of Buffer RW1 to an RNEasy spin column.</li>
<li>Centrifuge the sample at 8,000 x g for 1 minute at room temperature and then discard the flow through.</li>
<li>DNAse treatment with Qiagen RNAse-Free DNAse Kit: DNAse treatment is optional but is highly recommended for the highest quality samples.
<ol style="list-style-type:upper-alpha;">
<li>When you first receive the DNAse treatment kit, you will need to prepare the DNAse I stock solution by adding 550 &mu;l nuclease-free water to the DNAse. Mix gently by inverting and do not vortex. Aliquot into single use vials containing 10 &mu;l of the stock solution. You can store the stock solution at -20&deg;C for up to 9 months. The thawed aliquots can be stored at 4&deg;C for 6 weeks if needed, but do not refreeze the aliquots after thawing.</li>
<li>To prepare the DNAse I incubation mix add 70 &mu;l Buffer RDD to 10 &mu;l DNAse I stock solution per sample. Mix by gentle inversion and centrifuge tube briefly to collect residual liquid form the sides of the tube. Add 80 &mu;l DNAse I incubation mix directly to the RNEasy spin column. Incubate the DNAse treatment for 30 minutes at room temperature.</li>
</ol></li>
<li>Following DNAse treatment add 350 &mu;l Buffer RW1 to the spin column and centrifuge at 8,000 x g for 1 minute at room temperature.</li>
<li>After centrifugation discard the flow through and add 500 &mu;l Buffer RPE to the spin column. Incubate sample for 5 minutes at room temperature.</li>
<li>Centrifuge sample at 8,000 x g for 1 minute at room temperature and discard the flow through.</li>
<li>After centrifugation add 500 &mu;l 75% ethanol to the spin column.</li>
<li>Repeat centrifugation at 8,000 x g, but for 2 minutes at room temperature.</li>
<li>After centrifugation discard the flow through and allow the column to dry for 5 minutes.</li>
<li>Immediately centrifuge at 8,000 x g for 5 minutes at room temperature to remove any left over traces of ethanol.</li>
<li>After centrifugation is complete transfer the spin column into a new 1.5 ml collection tube and add 10 &mu;l nuclease-free water. Incubate for 1 minute and then centrifuge at 10,000 x g for 1 minute at room temperature. The RNA will be in the flow through.</li>
<li>Add another 10 &mu;l of nuclease-free water to the spin column and incubate at room temperature for 1 minute. Centrifuge sample at 10,000 x g for 1 minute at room temperature.</li>
<li>After centrifugation remove and discard the spin column. RNA will be in the flow through.</li>
<li>Check the quantity and quality of the RNA using a NanoDrop ND-1000 spectrophotometer following the manufacturer&#x2019;s instructions.</li>
<li>In addition, run a denaturing gel or use an Agilent 2100 bioanalyzer to check the integrity of the RNA.</li>
<li>Samples should be stored at -80&deg;C until cDNA synthesis can be performed.</li>
</ol>
Part 3: cDNA Synthesis using the Invitrogen SuperScript First-Strand System
RNA is easily prone to cleavage by RNAses leading to degradation. As a result many gene expression protocols have been developed to use a more stable cDNA product that has been directly synthesized from the RNA. Here we detail synthesis of first-strand cDNA using the Invitrogen SuperScript First-Strand Synthesis System3. Each cDNA synthesis reaction can accommodate up to 5 &mu;g of RNA. 
<ol>
<li>Before beginning the procedure mix and briefly centrifuge each component.</li>
<li>Prepare RNA/primer mixture in a sterile 0.5 ml microfuge tube as outlined in Table 1.</li>
<li>Incubate the sample at 65&deg;C for 5 minutes.</li>
<li>Immediately transfer sample to ice slurry for 10 minutes.</li>
<li>While the sample is cooling prepare a master mix with the components outlined in Table 2. The volumes are listed per 1 reaction. Adjust volumes accordingly for the total number of reactions.</li>
<li>After cooling, add 9 &mu;l of the master mix prepared in step 5 to each RNA/primer mixture. Mix gently and collect by brief centrifugation. The total volume of each sample should be 19 &mu;l.</li>
<li>Incubate sample at 42&deg;C for 2 minutes in a thermocycler.</li>
<li>Add 1 &mu;l (50 units) of SuperScript II RT to each tube, mix, and incubate at 42&deg;C for 60 minutes in a thermocycler.</li>
<li>Terminate the reaction at 70&deg;C for 15 minutes and then chill sample to 4&deg;C. A thermocycler can be programmed for steps 8 and 9.</li>
<li>After the sample has reached 4&deg;C, transfer the reaction to an ice bath.</li>
<li>On ice add 1 &mu;l of RNAse H to each tube and incubate for 20 minutes at 37&deg;C. The total volume is now 21 &mu;l. Addition of RNAse H to your sample will degrade the RNA template and leave only the single strand cDNA product.</li>
</ol>
Part 4: cDNA Isolation and Precipitation
<ol>
<li>A 1.5 ml phase lock gel tube will be used in the isolation of the cDNA product. Prepare a 1.5 ml phase lock gel tube by centrifugation at 12,000 x g for 2 minutes. The gel should all be at the bottom of the tube.</li>
<li>Add 81.5 &mu;l phenol (Tris-saturated, buffered to pH 8.0), 81.5 &mu;l of 24:1 chloroform isoamyl alcohol, and the sample (21 &mu;l) into the phase lock gel tube. Mix several times by gentle inversion.</li>
<li>Centrifuge the sample at 12,000 x g for 5 minutes at room temperature.</li>
<li>The cDNA should be in the upper aqueous phase. Transfer the upper phase into a clean 1.5 ml microfuge tube. The total volume should be around 20 &mu;l.</li>
<li>Add 1X volume of 3 M NaOAc, pH 5.2 and mix gently. For example, if the volume of the upper aqueous phase was equal to 20 &mu;l, you will need to add 2 &mu;l of 3 M NaOAc, pH 5.2.</li>
<li>Add 7 &mu;l of 5 mg/ml glycogen and mix gently.</li>
<li>Add 1X volume of isopropanol and mix gently. Generally this is around 30 &mu;l of isopropanol.</li>
<li>Allow sample to incubate at room temperature for 10 minutes and then centrifuge at 12,000 x g for 20 minutes at room temperature.</li>
<li>A small cDNA pellet should form on the bottom of the tube. Carefully remove the supernatant with a pipette.</li>
<li>Add 500 &mu;l of 70% ethanol and mix by inversion.</li>
<li>Centrifuge the sample at 12,000 x g for 5 minutes.</li>
<li>After centrifugation remove the supernatant with a pipette and repeat the 70% ethanol wash and centrifugation (steps 10 and 11).</li>
<li>After centrifugation remove the supernatant with a pipette. If there is liquid remaining on the sides of the tube, it can be gathered by briefly spinning the tube. Remove any excess liquid and allow the pellet to dry at room temperature for 5 minutes.</li>
<li>Add 20 &mu;l of nuclease-free water to rehydrate the pellet. Place the sample at 55&deg;C for 5 minutes for rehydration of the pellet.</li>
<li>The quantity and quality of the cDNA product is then checked using a NanoDrop ND-1000 spectrophotometer following the manufacturer&#x2019;s instructions. The cDNA can be stored at -20&deg;C.</li>
</ol>
Representative Results 
After RNA cleanup, approximately 15 &mu;g of high quality total RNA can be expected from 50 zebrafish embryos. To assess total RNA quality, a spectrophotometer, gel electrophoresis, and an Agilent 2100 Bioanalyzer can be used. A NanoDrop ND-1000 spectrophotometer can be used to assess the absorbance at 260 nm in comparison to the absorbance at 280 nm (Figure 1). The A260/A280 ratio can reveal the presence of contaminants and give evidence of possible degradation. An A260/A280 ratio of 1.9-2.0 is considered acceptable. In addition, it is highly recommended that an RNA denaturation gel be run to assess RNA integrity. RNA strands will separate on basis of their size, with smaller strands migrating further down the gel. A non-degraded sample will appear as a smear with two strong bands. The smear is a result of a population of different sized mRNA strands and the bands correspond to 28S and 18S rRNA. The 28S band should be about twice as intense as the 18S band (Figure 2). Alternatively, total RNA integrity can be assessed using an Agilent 2100 bioanalyzer. Two distinct peaks representing the 28S and 18S rRNA is expected (Figure 3). The 28S peak should be larger than the 18S peak with the area under the peaks approximately 2:1, respectively. Two distinct peaks will not be observed in degraded samples. RNA samples exhibiting degradation should not be carried through subsequent experimental procedures.
Spectrophotometric analysis can be used to assess the quality of the cDNA product (Figure 4). The A260/A280 should be around 1.8. cDNA reactions with an input of 5 &mu;g of total RNA routinely yield 1-2 &mu;g of cDNA product in our laboratory.
<img src="/files/ftp_upload/1470/1470fig1.jpg" alt="Figure 1" /> Figure 1. A NanoDrop spectrum of RNA sample. After RNA cleanup, RNA quantity and quality can be assessed using a NanoDrop ND-1000 spectrophotometer. Approximately 15 &mu;g of high quality total RNA is routinely yielded from 50 zebrafish embryos with an A260/A280 ratio around 1.9-2.0.
<img src="/files/ftp_upload/1470/1470fig2.jpg" alt="Figure 2" /> Figure 2. An RNA denaturation gel. To assess the integrity of the total RNA isolation, an RNA denaturation gel can be ran. A smear with two bright bands corresponding to 28S and 18S rRNA is expected. The 28S band should be approximately twice as intense as the 18S band.
<img src="/files/ftp_upload/1470/1470fig3.jpg" alt="Figure 3" /> Figure 3. A trace of an RNA sample from a bioanalyzer. To assess the integrity of the total RNA isolation, samples may also be analyzed on an Agilent 2100 bioanalyzer. Two sharp peaks, corresponding to the 28S and 18S rRNA, should be visible. The 28S peak should be larger than the 18S peak with the area under the peaks approximately 2:1, respectively.
<img src="/files/ftp_upload/1470/1470fig4.jpg" alt="Figure 4" /> Figure 4. A NanoDrop spectrum of a cDNA sample. Spectrophotometric analysis with a NanoDrop ND-1000 can be used to assess the quantity and quality of the cDNA product. The A260/A280 ratio should be around 1.8. cDNA reactions with an input of 5 ug of total RNA routinely yield 1-2 ug of cDNA in our laboratory.
Table 1. Components to add for RNA/primer mixtures at step 2 of cDNA synthesis.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>Total RNA (up to 5 &mu;g)</td>
 <td>10 &mu;l</td>
 </tr>
 <tr>
 <td>10 mM dNTP mix</td>
 <td>1 &mu;l</td>
 </tr>
 <tr>
 <td>Oligo(dT)12-18 (0.5 &mu;g/ &mu;l)</td>
 <td>1 &mu;l</td>
 </tr>
 <tr>
 <td>DEPC-treated water</td>
 <td>n &mu;l</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>10 &mu;l</td>
 </tr>
</tbody>
</table>
Table 2. Reaction mixture for step 5 of cDNA synthesis. Volumes listed are per reaction. Increase volumes of each component for the total number of reactions.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 </tr>
 <tr>
 <td>10X RT Buffer</td>
 <td>2 &mu;l</td>
 </tr>
 <tr>
 <td>25 mM MgCl2</td>
 <td>4 &mu;l</td>
 </tr>
 <tr>
 <td>0.1 mM DTT</td>
 <td>2 &mu;l</td>
 </tr>
 <tr>
 <td>RNAseOUT</td>
 <td>1 &mu;l</td>
 </tr>
</tbody>
</table>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> TRIzol Reagent Manual. , (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNEasy Mini Handbook. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SuperScript First-Strand Synthesis System for RT-PCR. , (2003).</li></ol>

The authors have nothing to disclose.

The fragility of the RNA molecule is the most critical consideration that should be remembered throughout this protocol. Sterile equipment that is RNAse-free should always be used in an RNAse-free zone of the laboratory. It is helpful to reserve a part of the laboratory to be used for RNA procedures only. This area should be frequently sprayed with an RNAse removing product, such as RNAse Away. The RNA samples should always be handled with gloves and kept on ice to prevent degradation.
This pr...

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<td>Glycogen (5 mg/ml)</td>
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rna isolation from embryonic zebrafish and cdna synthesis for gene expression analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

Watch this Scientific Journal Video about RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis at JoVE.com

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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37.7K Views.  Purdue University. The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Video: RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

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

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

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

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

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

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...