We thank Sacha Reichman and Dominique Santiard-Baron for their help in editing the RNA purification protocol.

1. jouRNAl: Procedure to Recover the Specimens from the Surgical Block
In the lab
<ol>
	<li>Get an importation contract from express shipping company.</li>
	<li>Fill 10 (or 25) shipping forms with the postal address of the laboratory indicating the contact person in the laboratory (Phone number and Email address).</li>
	<li>Prepare 10 (or 25) samples forms numbered 1 to 10 (or 25). These forms include dedicated spaces to inform on a) anonymous identification of the patient, b) the date of the surgery and c) any additional remarks the surgeon would like to add. Prepare 10 (or 25) padded envelopes (150 x 210 mm).</li>
	<li>Prepared using RNAse-free reagents 25 (or 65) ml of 6 M guanidine chloride in diethylpyrocarbonate (DEPC)-treated H2O (GHCl).</li>
	<li>Fill 10 or 25 numbered 5 ml sterile polyethylene round bottom tubes with 2.4 ml of GHCl solution.</li>
	<li>Used argon gas to fill the top part of these tubes, than press tightly on the cap to prevent the oxidation of the GHCl solution over several years of storage in the surgical cabinet.</li>
	<li>Introduce every 5 ml tubes in a 50 ml sterile polypropylene conical bottom tube with screw cap. Use a piece of clean paper tissue to hold the 5 ml tube into the 50 ml tube, close the tube.</li>
	<li>Place the 50 ml tubes on a polystyrene rack and the rack into a cardboard box with the padded envelopes, the shipping forms, the samples forms.</li>
	<li>Paste the instructions (see : 1.12-1.19) inside of the cover of the cardboard box to facilitate their reading.</li>
	<li>Send the cardboard box by mail to the contact person in the hospital.</li>
</ol>
In the hospital
<ol start="11">
	<li>Bring the cardboard box from the surgical cabinet to the surgical room. Caution note: if the procedure involves an immune compromised patient, the cardboard should not be brought to the surgical room.</li>
	<li>During the surgery, place the retinal specimen into numbered 5 ml sterile polypropylene filled with GHCl solution. Close tightly the tube.</li>
	<li>Return the tube briefly.</li>
	<li>Place the tube on the blood tube rocker for 10 min.</li>
	<li>Fill in the accompanying sample numbered form.</li>
	<li>Report the identification number onto the corresponding numbered 50 ml tube.</li>
	<li>Introduce the 5 ml tube with the surgical specimen into the 50 ml tube, replace paper tissue and screw the tube.</li>
	<li>Introduce the tube and the filled sample form into it in one of the accompanying padded envelopes. Close it.</li>
	<li>Call the express shipping company for pick up.</li>
</ol>
2. RNA Purification
In the lab
<ol>
	<li>Homogenize the specimen in its 5 ml tube polypropylene with GHCl solution with a homogenizer for 1 min while moving the tube up and down.</li>
	<li>Add 270 &mu;l of 2 M potassium acetate pH 5.0. Shake vigorously for 10 min by placing the tube on a shaker in its horizontal position (420 min-1).</li>
	<li>Centrifuge 10 min at 5,000 rpm (6,500 x g) at 20 &deg;C.</li>
	<li>Aspirate smoothly the soluble fraction without disturbing the pellet.</li>
	<li>Transfer into a 14 ml sterile tube.</li>
	<li>Add 5.3 ml of 100 mM Tris pH 8.0, N-Lauroylsarcosine 1%.</li>
	<li>Add 3.2 g of cesium chloride (CsCl) and mix the tube by vortexing.</li>
	<li>Add 1.8 ml of CsCl/ ethylenediaminetetraacetic acid (EDTA) in a sterile 11 ml polyallomer centrifuge tube.</li>
	<li>With a 10 ml sterile Pasteur pipette, transfer the RNA solution onto 1.8 ml CsCl/EDTA by sliding slowly on the edge of the tube to avoid disturbing the density cushion.</li>
	<li>Place the tubes (a second tube containing the buffers without retina if necessary) into the rotor.</li>
	<li>Centrifuge 24 hr at 32,000 rpm (225,000 x g) at 20 &deg;C.</li>
	<li>Remove the superior part of the solution with a sterile Pasteur pipette, and discard it.</li>
	<li>Remove gradually while checking the moment when the DNA (viscous) is aspirated with a second sterile Pasteur pipette, and discard it.</li>
	<li>Remove the remaining solution taking care not to release the RNA pellet with a third sterile Pasteur pipette.</li>
	<li>Section the bottom of the tube with a scalpel flame-sterilized, then put the remaining part of the tube it upside down on a sterile gaze.</li>
	<li>Reverse the tube and rinse delicately with 160 &mu;l of GHCl.</li>
	<li>Let the pellet dry for 10 min.</li>
	<li>Resuspend the pellet in 150 &mu;l of (10 mM Tris pH 7.5 - 1 mM EDTA - 0.1% SDS).</li>
	<li>Transfer the solution into a 2 ml sterile microcentrifuge tube, then harvest the residual pellet with 30 &mu;l of (10 mM Tris pH 7.5 - 1 mM EDTA - 0.1% SDS).</li>
	<li>Add 150 &mu;l of (10 mM Tris pH 7.5 - 1 mM EDTA).</li>
	<li>Add 30 &mu;l of 3 M sodium acetate pH 5.0, vortex the tube.</li>
	<li>Add 900 &mu;l of ethanol 100% (-20 &deg;C), vortex the tube.</li>
	<li>Place the tube 30 min in melting ice.</li>
	<li>Centrifuge the tube 30 min at 15,000 rpm at 4 &deg;C.</li>
	<li>Aspirate delicately the soluble fraction, and discard it.</li>
	<li>Add 500 &mu;l 70% ethanol (RT), vortex the tube.</li>
	<li>Centrifuge the tube 20 min at 15,000 rpm (18,000 x g) at 4 &deg;C.</li>
	<li>Repeat the rinsing step (70% ethanol).</li>
	<li>Centrifuge briefly and eliminate the remaining ethanol with a P200 pipette.</li>
	<li>Let the pellet air dry for 10 min.</li>
	<li>Resuspend the pellet in 50 &mu;l DEPC-treated H2O.</li>
	<li>Mix vigorously by vortexing.</li>
	<li>Incubate 15 min at 45 &deg;C in a water bath.</li>
</ol>
3. RNA Analysis by Gel Electrophoresis
<ol>
	<li>Pour an agarose gel inside a chemical hood.</li>
	<li>In a sterile 1.5 ml microcentrifuge tube, add 2 &mu;l of RNA to be analyzed and 6.4 &mu;l of sample prep buffer.</li>
	<li>In a second 1.5 ml tube, add 3 &mu;l of RNA standards and 9.6 &mu;l of sample prep buffer.</li>
	<li>Heat the tubes 15 min at 65 &deg;C, put them on ice.</li>
	<li>Add 1 &mu;l ethidium bromide (EB) loading buffer without dye in the RNA sample tube and 1 &mu;l EB loading buffer with dye in the RNA standards tube.</li>
	<li>Run the gel under 80 - 100 V Inside a chemical hood in running buffer, until one of the dyes (bromophenol blue) reaches 2/3 of the bottom of the gel.</li>
	<li>Rinse the gel twice 15 min with 250 ml de DEPC-treated 2x SSC.</li>
	<li>Take a digitalized image under UV illumination.</li>
	<li>Calculate the ratio between the upper band (23S rRNA) and the lower band (16S rRNA).</li>
</ol>
4. Final Purification of the RNA
<ol>
	<li>Adjust the volume of the RNA sample to 100 &mu;l with DEPC-treated H2O (if necessary).</li>
	<li>Add 100 &mu;l of Acid phenol (1 ml phenol saturated in DEPC-treated H2O + 130 &mu;l of 50 mM of sodium acetate, pH 5.2), vortex vigorously.</li>
	<li>Centrifuge the 10 min at 13,000 rpm (15,000 x g) at room temperature.</li>
	<li>Recover carefully and transfer the aqueous phase (upper phase) into a novel a sterile 1.5 ml microcentrifuge tube.</li>
	<li>Add 10 &mu;l of 3 M sodium acetate, pH 5.2 and 250 &mu;l of ethanol 100% (-20 &deg;C).</li>
	<li>Incubate the tube 30 min into melting ice.</li>
	<li>Centrifuge the tube 30 min at 14,000 rpm (18,000 x g) at 4 &deg;C.</li>
	<li>Aspirate carefully the soluble fraction, and discard it.</li>
	<li>Add 500 &mu;l of 70% ethanol (room temperature), and vortex the tube.</li>
	<li>Centrifuge the tube 20 min at 14,000 rpm (18,000 x g) at 4 &deg;C.</li>
	<li>Repeat the rinsing step (70% ethanol).</li>
	<li>Centrifuge briefly and eliminate the remaining ethanol with a P200 pipette.</li>
	<li>Let air dry the pellet for 10 min.</li>
	<li>Resuspend the pellet into 50 &mu;l of DEPC-treated H2O.</li>
	<li>Incubate then tube 15 min at 45 &deg;C.</li>
	<li>Measure the absorbance (Abs) at 260 nm and 280 nm on 2 &mu;l of the RNA sample. Calculated the ratio Abs260/Abs280.</li>
	<li>Store the RNA sample at -80 &deg;C (stable for several years).</li>
</ol>
5. Solutions and Cleaning Procedures
<ol>
	<li>Potassium acetate 2 M, pH 5.0: Dissolve 19.6 g Potassium acetate into 70 ml of DEPC-treated H2O. Clean the pH meter probe by soaking it into with 1M NaOH for 15 min, followed by 5 rinses with DEPC-treated H2O. Adjust to pH 5.0 with acetic acid then adjust volume to 100 ml with DEPC-treated H2O.</li>
	<li>Tris-HCl 100 mM, pH 8.0, N-Lauroylsarcosine 1%: Dissolve 2 g of N-Lauroylsarcosine (under a hood) in to 40 ml of 0.5 M Tris-base pH 8.0. Adjust volume to 200 ml with DEPC-treated H2O.) CsCl/EDTA: 96 g of CsCl in 50 ml 10 mM EDTA pH 7.5 prepared with DEPC-treated H2O. Autoclave and adjust volume to 100 ml with DEPC-treated H2O. Check for pH&lt;7.5.</li>
	<li>Agarose gel 1%: Put 1 g of agarose in 62 ml DEPC-treated H2O. Boil in a microwave oven. Add 18 ml of 12.3 M formaldehyde, 20 ml of 5x MOPS. Pour inside a chemical hood.</li>
	<li>Sample prep Buffer: To be prepared extemporary. Mix inside a chemical hood 8 &mu;l of 5x MOPS, 16 &mu;l of 12.3 M formaldehyde and 40 &mu;l of formamide.</li>
	<li>EB loading buffer (without dye): To be prepared extemporary. Add 4 &mu;l 10 mg/ml ethidium bromide into 20 &mu;l of 80% glycerol prepared with DEPC-treated H2O.</li>
	<li>EB loading buffer (with dye): To be prepared extemporary. Add 2 &mu;l 10 mg/ml ethidium bromide into 10 &mu;l of 80% glycerol containing 0.25% xylen cyanol, 0.25% bromophenol blue with DEPC-treated H2O.</li>
	<li>Running buffer: To 60 ml 5x MOPS, add 54 ml of 12.3 M formaldehyde and 186 ml of DEPC-treated H2O.</li>
	<li>DEPC-treated H2O: Incubate distilled water with 0.1% v/v diethylpyrocarbonate for 2 hr at 37 &deg;C with stirring. Sterilize by Autoclave.</li>
	<li>Cleaning the homogenizer: Add 25 ml of cleaning solution (RBS) to 1 L of DEPC-treated H2O, and immerge the axis 1 min with stirring. Incubate 2 hr at 60 &deg;C. Rinse thoroughly with DEPC-treated H2O Wrap the instrument into aluminum and put into the instrument box. Wrap the box. Sterilize by Autoclave.</li>
	<li>Cleaning the electrophoresis device: Wash the apparatus, the tray and the 15-wells comb. Incubate 15 min in 3% hydrogen peroxide. Rinse once with 100% ethanol. Air dry.</li>
	<li>Cleaning the glass dishes: Clean the glass dishes RBS 2% all the night, then rinse with distilled water before sterilization at 200 &deg;C.</li>
</ol>

<ol>
	<li>Mitry, D., Charteris, D. G., 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=The+epidemiology+of+rhegmatogenous+retinal+detachment:+geographical+variation+and+clinical+associations.">The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations.</a> The British Journal of Ophthalmology. 94 (6), 678 (2010).</li><li>Green, R. E., Krause, J., 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=A+draft+sequence+of+the+Neandertal+genome.">A draft sequence of the Neandertal genome.</a> Science. 328 (5979), 710 (2010).</li><li>Glisin, V., Crkvenjakov, R., 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=Ribonucleic+acid+isolated+by+cesium+chloride+centrifugation.">Ribonucleic acid isolated by cesium chloride centrifugation.</a> Biochemistry. 13 (12), 2633 (1974).</li><li>Van Gelder, R. N., von Zastrow, M. 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=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).</li><li>Cavalieri, L. F., Yamaura, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+tRNAs+as+inhibitors+of+viral+reverse+transcription+in+vitro.">coli tRNAs as inhibitors of viral reverse transcription in vitro.</a> Nucleic Acids Research. 2 (12), 2315 (1975).</li><li>Sawadogo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+inhibition+of+yeast+RNA+polymerases+A+and+B+by+tRNA+and+alpha-amanitin.">On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin.</a> Biochemical and biophysical research communications. 98 (1), 261 (1981).</li><li>Delyfer, M. N., Raffelsberger, W., 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=Transcriptomic+analysis+of+human+retinal+detachment+reveals+both+inflammatory+response+and+photoreceptor+death.">Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.</a> PloS ONE. 6 (12), e28791 (2011).</li><li>Kalathur, R. K., Gagniere, N., 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=RETINOBASE:+a+web+database,+data+mining+and+analysis+platform+for+gene+expression+data+on+retina.">RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina.</a> BMC Genomics. 9, 208 (2008).</li><li>Nakazawa, T., Hisatomi, T., 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=Monocyte+chemoattractant+protein+1+mediates+retinal+detachment-induced+photoreceptor+apoptosis.">Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).</li><li>Leveillard, T., Sahel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod-derived+cone+viability+factor+for+treating+blinding+diseases:+from+clinic+to+redox+signaling.">Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling.</a> Science Translational Medicine. 2 (26), 26ps16 (2010).</li><li>Chalmel, F., Leveillard, T., 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=Rod-derived+Cone+Viability+Factor-2+is+a+novel+bifunctional-thioredoxin-like+protein+with+therapeutic+potential.">Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential.</a> BMC Molecular Biology. 8, 74 (2007).</li></ol>

The authors have nothing to disclose.

The development of a procedure for tissue recovery from the surgical block has been essential to the transcriptome analysis of retinal detachment. One should notice that this type of surgery is practiced in emergency and that the ophthalmologists operating have little time to participate in a biological research program when they operate. This retinectomy is also performed stochastically in each service, so that the easier way to reach statistical numbers is to work with a network. In such network, the standardiza...

The main therapeutic objective in retinal detachment (RD) is to find a way to limit photoreceptor cell damage and retinal inflammation resulting from the separation of the photoreceptors from the retinal pigmented epithelial cells. During RD, RPE cells are activated, migrate, dedifferentiate, and proliferate at the surface of the detached retina, exerting contractile forces leading to complications. Transcriptomics analysis of RD is a way to identify target genes with modified expression following RD and hence future therapeutic molecules that could improve final visual outcome in combination with surgery. It is well known ribonucleic acid (RNA) is not stable as is deoxyribonucleic acid (DNA), the latter being used extensively for genetic studies, its stability had permitted the sequencing of the Neanderthal genome from paleontological samples of more than 30,000 years old 2. Transcription of DNA of the genes from the genome into messenger RNA is the major process in gene expression, and the mRNA is labile in order to constitute a signal. RNAs are very rapidly degraded by RNAse enzymes which terminate the signal. When tissues are isolated from an organism, the RNAs are very often degraded before the expression is studied in a research laboratory. Degraded RNA is not suited for analysis gene expression. Because the laboratory staff cannot participate in surgery, we developed a procedure that is easy and only requires that the surgeon to recover the tissues into an appropriate RNAse-free solution. The RNA of the tissues is stable and can be analyzed without any sign of degradation after 72 hr at room temperature in this solution. The RNAs from specimens are purified by a standardized method involving an ultracentrifugation on a cesium chloride gradient after having been transferred to the laboratory 3. Then, quality of the RNAs are assessed by agarose gel electrophoresis and by RT-PCR. The RNA purification protocol has the advantage of separating the molecules according to their density that is different for DNA and RNA and assures that the RNA is not contaminated by a DNA molecules that will generate artifactual signals in gene expression studies. In addition, the transfer RNAs (tRNAs), which are quantitatively the most abundant RNA within a cell, are separated according to the same physical property from the ribosomal RNA (rRNAs) and the messenger RNAs (mRNAs), those two last one being the end product of the purification process. The removal of tRNA from the preparation is useful since most of the microarray analytical protocols involved the use of reverse transcriptases and RNA polymerases which are inhibited by tRNA 4-6. The purified RNA from surgical specimens are labeled using standard protocol and hybridized to a microarray chip and the results are analyzed using two complementary methods, the false discovery rate method, and using a novel method based on mutual information and visualized on the web-based server Retinobase 7,8.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Aventi J-E</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>JS-5.3</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 <td>Beckman Coulter</td>
 <td>LE 80K</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotor</td>
 <td>Beckman Coulter</td>
 <td>SW41</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>Biorad</td>
 <td>Pac 3000</td>
 <td></td>
 </tr>
 <tr>
 <td>PolytronTM</td>
 <td>Kinematica</td>
 <td>PT 2100</td>
 <td>Supplied with PT-DA 2105:2</td>
 </tr>
 <tr>
 <td>Agarose gel electrophoresis device</td>
 <td>Biorad</td>
 <td>MiniGel Cell GT</td>
 <td></td>
 </tr>
 <tr>
 <td>Imaging System </td>
 <td>Biorad</td>
 <td>GelDoc-It Imager</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml sterile polyethylene tube</td>
 <td>Greiner</td>
 <td>115261</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile polyallomer centrifuge tube </td>
 <td>Beckman Coulter</td>
 <td>331372</td>
 <td></td>
 </tr>
 <tr>
 <td>Chemical reagents</td>
 <td>Sigma</td>
 <td></td>
 <td>Molecular biology grade (RNase and DNase free products)</td>
 </tr>
 <tr>
 <td>Cleaning Solution </td>
 <td>VWR </td>
 <td>RBS</td>
 <td></td>
 </tr>
 <tr>
 <td>Microarray chip</td>
 <td>Affymetrix</td>
 <td>Human U133 plus 2 array</td>
 <td></td>
 </tr>
 </tbody>
</table>

transcriptomic analysis of human retinal surgical specimens using journal

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

The procedure jouRNAl (Figure 1) allows us recover specimens of retina from the surgical block to purified RNA, and to analyze the transcriptome of retinal detachment. Retinal detachment results in the upregulation of the monocyte chemotactic MCP1 gene CCL2, and in the decrease in expression of the rod photoreceptor transducing gene GNAT1, the short wave cone opsin OPN1SW, and the homeogene CRX (Figure 2). The induction of CCL2 is resultin...

Watch this Scientific Journal Video about Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl at JoVE.com

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

transcriptomic-analysis-human-retinal-surgical-specimens-using

Research

JoVE Journal

Medicine

7.7K visualizações.  Institut National de la Santé et de la Recherche Médicale. Usamos amostras de retina de retinectomy para uma análise transcriptomic de descolamento de retina. Foi desenvolvido um processo que permite a conservação de ARN entre os blocos cirúrgicos e laboratoriais. Temos um protocolo padronizado para purificar RNA por ultracentrifugação de cloreto de césio para assegurar que os ARN purificados são adequados para a análise de microarray.

Vídeo: Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Medicina

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocolo melhorado para microdissecção a laser de ilhotas pancreáticas humanas a partir de espécimes cirúrgicos

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Dois métodos para estabelecer células endometriais Humano estromais primários de espécimes de histerectomia

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

A Passo a Passo Protocolo de Cirurgia sub-retiniana em Coelhos

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

Geração de um humanizado rato Liver Usando células-tronco hepáticas humanas

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

A microscopia intravital da vasculatura Tumor-associado usando avançada Dorsal câmaras de janela dobras cutâneas em ratos transgênicos fluorescentes

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Usando em Vitro viver-pilha de imagem para explorar Chemotherapeutics entregues por nanopartículas de lipídios-baseado

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

Treinamento cirúrgico para implantação de microeletrodos Neocortical matrizes usando um modelo de cadáver humano formaldeído-fixo

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Diagnosis and Surgical Treatment of Human Brucellar Spondylodiscitis

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in pastoral communities. It is difficult to differentially diagnose and usually leads to irreversible neurologic deficits and spinal deformities. The initial diagnosis of BS is based on clinical findings and radiographic assessments, and the confirmed diagnosis should be established by the isolation of Brucella species from the blood and/or the standard tube agglutination test. Differential diagnosis of multifocal BS from either degenerative disc diseases or tuberculosis is especially highlighted. The surgical approach, either endoscopic or open, is demonstrated in detail, accompanied by radiographic evidence of structural compression or severe instability. Further, the crucial surgical steps, including single-stage transforaminal decompression, debridement, interbody fusion, and internal fixation, are explained. Moreover, perioperative care and postoperative rehabilitation are also addressed. Taken together, this clinical algorithm presents a practical guide that has yielded substantially satisfactory outcomes in the past decades, which can also be introduced for large-scale application to manage human BS, especially in endemic regions.

We describe a clinical algorithm, based on decades of front-line experience of diagnosis and surgical treatment of human Brucellar spondylodiscitis in the largest medical center of the China's Xinjiang Pastoral Area.

Diagnóstico e Tratamento Cirúrgico da Espondilodiscite Humana de Brucellar

Brucellar spondylodiscitis (BS) is the most prevalent and significant osteoarticular presentation of human Brucellosis, which is commonly manifested in ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Indução e Análise do Estresse Oxidativo em Células Epiteliais de Pigmento Epitelial Humano Transposon-Transfected Da Bela Adormecida

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...