Le projet décrit a été soutenue par Prix Nombre R01MH079751 (PI: F. Peruzzi) de l&#39;Institut national de la santé mentale. Le contenu est exclusivement la responsabilité de leurs auteurs et ne représentent pas nécessairement les vues officielles de l&#39;Institut national de santé mentale ou l&#39;Institut national de la santé.

<ol>
	<li>Pacifici, M., 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=Cerebrospinal+fluid+miRNA+profile+in+HIV-encephalitis.">Cerebrospinal fluid miRNA profile in HIV-encephalitis.</a> J. Cell. Physiol. 10, (2012).</li><li>Krol, J., Loedige, I., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+widespread+regulation+of+microRNA+biogenesis,+function+and+decay.">The widespread regulation of microRNA biogenesis, function and decay.</a> Nat. Rev. Genet.. 11, 597-610 (2010).</li><li>Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+signatures+in+human+cancers.">MicroRNA signatures in human cancers.</a> Nat. Rev. Cancer. 6, 857-866 (2006).</li><li>Chen, X., 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=Characterization+of+microRNAs+in+serum:+a+novel+class+of+biomarkers+for+diagnosis+of+cancer+and+other+diseases.">Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.</a> Cell Res. 18, 997-1006 (2008).</li><li>De Smaele, E., Ferretti, E., Gulino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+biomarkers+for+CNS+cancer+and+other+disorders.">MicroRNAs as biomarkers for CNS cancer and other disorders.</a> Brain Res. 1338, 100-111 (2010).</li><li>Heneghan, H. M., Miller, N., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNAs+as+biomarkers+and+therapeutic+targets+in+cancer.">MiRNAs as biomarkers and therapeutic targets in cancer.</a> Curr. Opin. Pharmacol. 10, 543-550 (2010).</li><li>Kosaka, N., Iguchi, H., Ochiya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNA+in+body+fluid:+a+new+potential+biomarker+for+cancer+diagnosis+and+prognosis.">Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis.</a> Cancer Sci. 101, 2087-2092 (2010).</li><li>Lu, 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=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li><li>Shah, A. A., Leidinger, P., Blin, N., Meese, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA:+small+molecules+as+potential+novel+biomarkers+in+cancer.">miRNA: small molecules as potential novel biomarkers in cancer.</a> Curr. Med. Chem. 17, 4427-4432 (2010).</li><li>Blondal, 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=Assessing+sample+and+miRNA+profile+quality+in+serum+and+plasma+or+other+biofluids.">Assessing sample and miRNA profile quality in serum and plasma or other biofluids.</a> Methods. 59, 1-6 (2013).</li><li>Jensen, S. 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=Evaluation+of+two+commercial+global+miRNA+expression+profiling+platforms+for+detection+of+less+abundant+miRNAs.">Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs.</a> BMC Genomics. 12, 435 (2011).</li></ol>

Les auteurs n&#39;ont rien à révéler.

Les microARN sont de petits ARN non codantes qui régulent l&#39;expression des gènes par l&#39;inhibition de la traduction et / ou de promotion de la dégradation de l&#39;ARNm 2. En raison de leur grande stabilité dans des conditions exemptes de cellules, les microARN ont été détectés dans de nombreux fluides corporels. En outre, le profil d&#39;expression de microARN distinct a été corrélée avec le stade et / ou de la progression dans une variété de cancers chez l&#39;homme 9.3. ...

Les microARN appartiennent à la famille de la petite (21-23 nt de long) non codantes des ARN qui régulent l&#39;expression des gènes post-transcriptionnel. microARN peuvent être sécrétées dans l&#39;espace extracellulaire où ils semblent être relativement stables. Alors que la détermination des changements dans l&#39;expression des miARN peut être une étape importante vers l&#39;identification de biomarqueurs, effectuer profils miRNA et la manipulation d&#39;une grande quantité de données peut être intimidant. Ici, nous décrivons un protocole pour déterminer les changements dans l&#39;expression des miARN dans le liquide céphalo-rachidien (applicable à d&#39;autres fluides corporels) par un temps réel sensible PCR. Nous décrivons également l&#39;utilisation de logiciels d&#39;analyse de données, y compris l&#39;analyse statistique et la représentation graphique des résultats. Toute la procédure est relativement simple et facile et, en fonction du nombre d&#39;échantillons à profilés et le nombre de machines de PCR en temps réel disponibles, également relativement rapides. La partie expérimentale nécessite précision dans la manipulationARN et pipetage dans des plaques à 384 puits, tandis que la section d&#39;analyse de données à l&#39;aide GenEx nécessite quelques connaissances de base en informatique et en statistiques.

Table III. List of equipment
<table border="1" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:27px;">
			Equipment
			</td>
			<td nowrap="nowrap" style="width:150px;height:27px;">
			Company
			</td>
			<td nowrap="nowrap" style="width:204px;height:27px;">
			Catalog Number
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Finnpipette Novus Multichannel Pipetter
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			HH05279
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Finntip Pipette Tips
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			9400613
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Thermal Cycler C1000
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			0.2 ml Low Profile, Clear PCR tubes
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			TLS0801
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Flat Cap Strips
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Biorad
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			TLS0803
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			LightCycler&#174; 480 Real-Time PCR System
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Roche
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			LightCycler&#174; 480 Sealing Foil
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Roche
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			04 729 757 001
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Refrigerated Centrifuge 5804R
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Swing-bucket Rotor, 4-place
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			A-4-44
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Mini Centrifuge
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			05-090-100
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Vortex
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			1.5 ml Microcentrifuge Tubes, Sterilized
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Fisher
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			02-681-5
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Matrix Reagent Reservoir, 25 ml
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Thermo Scientific
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			8093
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Thermomixer Confort
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Eppendorf
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-To Mini Centrifuge Sigma 1-15
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Sigma
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;"></td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:301px;height:21px;">
			Bench-Top Vortex
			</td>
			<td nowrap="nowrap" style="width:150px;height:21px;">
			Velp Scientifica
			</td>
			<td nowrap="nowrap" style="width:204px;height:21px;">
			F202A0173
			</td>
		</tr>
	</tbody>
</table>

Table IV. List of reagents
<table border="1" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<tbody>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:25px;">
			Reagents
			</td>
			<td nowrap="nowrap" style="width:168px;height:25px;">
			Company
			</td>
			<td nowrap="nowrap" style="width:144px;height:25px;">
			Catalog Number
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Universal c-DNA synthesis kit, 16-32 rxns
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203300
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			SYBR Green master mix, Universal RT, 25 ml
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203400
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Ultrapure Distilled Water Dnase, Rnase free
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Invitrogen
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			10977-015
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			MicroRNA Ready to use PCR Human Panels (I+II) V2.R
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Exiqon
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			203608
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			mirVana&#8482; miRNA isolation Kit, 40 isolations
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Ambion
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			AM1556
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			MS2 RNA carrier
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Roche Applied Science
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			10165948001
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			2Betamercaptoethanol 98%
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Sigma Aldrich
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			M3148-25ml
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Ethanol 100%
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Carlo Erba
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			64-17-5
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			Nuclease free water
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Qiagen
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			1039480
			</td>
		</tr>
		<tr>
			<td nowrap="nowrap" style="width:343px;height:21px;">
			RNAse Zap&#174;
			</td>
			<td nowrap="nowrap" style="width:168px;height:21px;">
			Ambion
			</td>
			<td nowrap="nowrap" style="width:144px;height:21px;">
			AM9780
			</td>
		</tr>
	</tbody>
</table>

cerebrospinal fluid microrna profiling using quantitative real time pcr

<html> Les microARN (miARN) constituent une couche puissante de la régulation des gènes en guidant RISC à cibler des sites situés sur des ARNm et, par conséquent, en modulant leur répression de la traduction. Changements dans l&#39;expression des miARN ont été révélés être impliqués dans le développement de toutes les grandes maladies complexes. En outre, des études récentes ont montré que les miARN peuvent être sécrétées dans l&#39;environnement extracellulaire et dans la circulation sanguine et d&#39;autres fluides corporels où ils peuvent circuler avec une grande stabilité. La fonction de ces miARN circulation reste largement insaisissable, mais des approches à haut débit systématiques, telles que miRNA tableaux de profilage, ont conduit à l&#39;identification de signatures miRNA dans plusieurs conditions pathologiques, y compris les maladies neurodégénératives et plusieurs types de cancers. Dans ce cadre, l&#39;identification de miARN profil d&#39;expression dans le liquide céphalo-rachidien, comme indiqué dans notre étude récente, rend miARN candidats intéressants pour l&#39;analyse de marqueurs biologiques. _content &quot;&gt; Il existe plusieurs outils disponibles pour le profilage de microARN, comme les puces, quantitative PCR en temps réel (qPCR), et le séquençage profond. Ici, nous décrivons une méthode sensible au profil microARN dans liquide céphalo-rachidien par PCR quantitative en temps réel. Nous utilisé les panneaux droits Exiqon microARN prêts à l&#39;emploi PCR I et II V2.R, qui permet la détection de 742 microARN humains uniques. Nous avons effectué les tableaux en triple courses et nous avons traité et analysé les données en utilisant le logiciel GenEx Professional 5. En utilisant ce protocole, nous avons profilé succès microARN dans divers types de lignées cellulaires et des cellules primaires, CSF, le plasma et les tissus inclus en paraffine fixés au formol.

Les résultats de cette étude ont été publiés 1. Figure 1 montre les résultats de l&#39;analyse des micro-ARN de référence de candidats en utilisant l&#39;application geNorm. En conséquence, deux microARN, miR-622 et miR-1266, ont été identifiés comme gènes de référence et ont été utilisés pour normaliser les valeurs de miARN. Pour l&#39;étude du LCR, nous avions trois groupes d&#39;échantillons: le VIH (n = 10), HIVE (n = 4), ...

Watch this Scientific Journal Video about Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR at JoVE.com

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Nous décrivons un protocole de PCR en temps réel pour le profil de microARN dans le liquide céphalo-rachidien (LCR). A l&#39;exception des protocoles d&#39;extraction d&#39;ARN, la procédure peut être étendue à l&#39;ARN extrait à partir d&#39;autres fluides corporels, des cellules en culture, ou des échantillons de tissus.

Liquide céphalo-rachidien profilage de microARN Utilisation quantitative PCR en temps réel

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

cerebrospinal-fluid-microrna-profiling-using-quantitative-real-time

Research

JoVE Journal

Medicine

15.3K vues.  LSU Health Sciences Center.  Nous décrivons un protocole de PCR en temps réel pour le profil de microARN dans le liquide céphalo-rachidien (LCR). A l'exception des protocoles d'extraction d'ARN, la procédure peut être étendue à l'ARN extrait à partir d'autres fluides corporels, des cellules en culture, ou des échantillons de tissus. 

Vidéo: Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Evaluation de la fixation des nanoparticules dans les tumeurs en temps réel par imagerie intravitale

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Recherche

Médecine

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Détection MicroRNA dans les tumeurs prostatiques par quantitative PCR en temps réel (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Mouvement recyclage en utilisant rétroaction en temps réel de la performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Utilisation quantitative PCR en temps réel pour déterminer prise de greffe de donneurs de cellules dans un modèle murin concurrentiel greffe de moelle osseuse

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

En temps réel cytotoxicité dans le sang total humain

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

Spectrométrie de masse quantitative de profilage Protéomes Cancer-cellulaires dérivées de liquide et de tumeurs solides

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

En temps réel d&#39;imagerie à rayons X du poumon Volumes fluide dans néonatale Souris Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Évaluation des métastases pulmonaires chez la souris tumeur mammaire modèles par PCR quantitative en temps réel

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

L&#39;isolement et le profilage des exosomes de Bile humain contenant microARN-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Time-lapse Extended Imagerie intravitale du temps réel multicellulaire Dynamics dans le microenvironnement de la tumeur

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...