This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).

HINWEIS: Vor der Bereitstellung Speichelproben alle Probanden gaben Einwilligung gemäß den Richtlinien für die Behandlung von Menschen bei Nationwide Kinderkrankenhaus. 1. Speichelentnahme und Lagerung <ol><li> Vor der Speichelentnahme, sicherzustellen, dass der Mund des Subjekts ist frei von Nahrung oder anderen Fremdstoffen, indem er das Thema spülen den Mund mit Wasser und die Vermeidung von Essen oder Trinken für 30 Minuten vor dem Sammeln der Probe. </li><li> Öffnen Sie ein 15 ml Zentrifugenröhrchen mit 2,5 ml DNA-Stabilisierungspuffer 2 achten Sie darauf, berühren Sie nicht die Innenseite der Kappe oder ein Rohr, und haben das Thema spucken 2,5 ml Speichel in die Pufferlösung. Hinweis: Das Sammeln von mehr als 2,5 ml Speichel können Probenabbau von einem unzureichenden Verhältnis von DNA-Probe, um Stabilisierungspuffer führen. Sammeln zu wenig Speichel erwarteten Erträge aus dem Protokoll zu reduzieren. Um Sammelmengen auswerten, die Ziffern Steigungen auf der Seite von Ter Rohr. </li><li> Die Schutzkappe und mischen durch Umdrehen, bis die Mischung homogenisiert. Heftiges Schütteln ist nicht notwendig. Lagern Sie die Proben bei Raumtemperatur für die kurzzeitige Lagerung oder 4 ° C für die Langzeitlagerung (&gt; 3 Monate). </li></ol> 2. Erste Vorbereitungen und Zell-Lyse <ol><li> Vor Beginn der Extraktion, Hitze ein Wasserbad auf 37 ° C, und bereiten einem Eimer Eis. Drei 15 ml konischen Zentrifugenröhrchen wird für jeden extrahierten Probe benötigt. Die drei Röhren wird verwendet, um die Zelle und Proteinpellet, die endgültige extrahiert gDNA und das Isopropanol und Ethanol-Überstände zu halten. </li><li> Abrufen Proben von Lagerung und Invertzucker Proben mehrmals dann Wirbel bei mittlerer Geschwindigkeit für 15 Sekunden. </li><li> Verzichten 2,5 ml Probe in ein sauberes 15 ml Zentrifugenröhrchen und 5 ml Zell-Lyse-Lösung. Die Probe 50-mal durch Inversion, und Inkubation bei RT für 30 min. </li></ol> 3. Entfernen RNA <ol><li> Dann werden 40 ul RNase A Solution bei 100 mg / ml, und bei 37 ° C für 15 min. </li><li> Entfernen Sie die Probe aus dem 37 ° C warmen Wasserbad und kühlen auf Eis für 3 min. <ol><li> Nach der RNase A Inkubation wird die Temperatur des Wasserbades auf 65 ° C für die DNA Rehydratisierung Schritt des Protokolls. </li></ol></li></ol> 4. Protein und Lipidentfernung <ol><li> Dann werden 50 ul Proteinase K-Lösung bei 20 mg / ml, Mischung mehrfach durch Umdrehen und inkubiert bei Raumtemperatur für mindestens 30 min. Hinweis: Dies ist eine mögliche Pause Punkt für das Protokoll. Nach der Zugabe der Proteinase-K-Lösung, kann die Probe bei 4 ° C gelagert, bis die Extraktion abgeschlossen werden kann. Lagerung bei 4 ° C für bis zu 24 Stunden wurde nicht gezeigt, um eine signifikante Wirkung auf die Extraktionsausbeuten oder DNA-Qualität haben. Langfristige Lagerung in diesem Stadium noch nicht bewertet worden. </li><li> Fügen Sie 1,7 ml Protein Precipitation Lösung, Vortex kräftig für 20 Sekunden bei hoher Geschwindigkeit und auf Eis für 10 min. </li&gt; <li> Nachdem die Proben auf Eis für 10 min, Zentrifuge für 10 min bei 3.000 × g und 4 ° C abgekühlt. Die gefällten Proteine ​​bilden muss eine enge Pellet, um fortzufahren. Wenn das Pellet ist nicht dicht oder die Lösung ist immer noch bewölkt, können die Proben auf Eis für 5 min mehr gekühlt werden und Zentrifugation wiederholt. Die Proben sind auf Eis gehalten werden, um eine enge Pellet gewährleisten. </li></ol> 5. Isolierung und Aufreinigung von gDNA <ol><li> In einen sauberen 15 ml-Zentrifugenröhrchen, Pipetten 5 ml Isopropanol und 8 ul Glycogen reinen Lösung bei 20 mg / ml. </li><li> Gießen der Überstand, der gDNA aus Schritt 4.3 in das Rohr mit dem Isopropanol und Glykogen Lösung, wobei hinter dem ausgefällten Proteinpellet. Nachdem der Überstand wurde hinzugefügt, vorsichtig mischen die Probe 50-mal durch Umdrehen und Zentrifuge für 30 min bei 3.000 × g und 4 ° C liegt. </li><li> Gießen Sie den Überstand langsam in einen sauberen 15-ml-Tube. Nach Entfernen des Überstands, Zugabe von 1 ml 70% EthanolWaschen des Pellets langsam und sanft schaukel Bewegen des Ethanol über dem ausgefällten Pellet mehrmals. Bewahren Sie das Ethanol in die Röhre. </li><li> Nach der ersten Wäsche, Zentrifugieren Sie die Probe für 1 min bei 2.000 xg und 20 ° C. Dieser Zentrifugationsschritt kann entweder bei 4 ° C oder 20 ° C durchgeführt werden. Keine signifikante Wirkung der Temperatur für diesen Schritt hat sich gezeigt. </li><li> Nach der ersten Wäsche und Zentrifugation des Pellets, gießen Sie langsam den Ethanolwasch aus der Tube und entsorgen, dann eine zweite Wasch durch Wiederholen der Schritte 5.3 und 5.4. </li><li> Nach dem Entfernen des Überstands aus der zweiten Wasch Pellet an der Luft trocknen lassen für 15 Minuten. <ol><li> Wenn die Probe nicht vollständig getrocknet ist, an der Luft trocknen für weitere 15 min. </li></ol></li></ol> 6. Rehydrierung von gDNA <ol><li> Nachdem die Probe getrocknet werden 300 ul Tris-EDTA, das getrocknete Pellet gDNA rehydrieren. </li><li> Vortex die Probe für 5 Sekunden bei mittlerer Geschwindigkeit und in einen65 ° C heißen Wasserbad für 1 Stunde. </li><li> Entfernen Sie die Proben aus dem Wasserbad inkubiert und O / N bei RT. HINWEIS: Alle Produkte und Reagenzien sind in der Tabelle aufgeführten Materialien, sowie Tabelle 4. </li></ol>

<ol>
	<li>Quinque, D., Kittler, R., Kayser, M., Stoneking, M., Nasidze, I. <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+saliva+as+a+source+of+human+DNA+for+population+and+association+studies.">Evaluation of saliva as a source of human DNA for population and association studies.</a> Analytical Biochemistry. 353, 272-277 (2006).</li><li>Min, J. L., 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=High+mircosatellite+and+SNP+genotyping+success+rates+established+in+a+large+number+of+genomic+DNA+samples+extracted+from+mouth+swabs+and+genotypes.">High mircosatellite and SNP genotyping success rates established in a large number of genomic DNA samples extracted from mouth swabs and genotypes.</a> Twin Research and Human Genetics. 9, 501-506 (2006).</li><li>Dlugos, D. J., Scattergood, T. M., Ferraro, T. N., Berrettinni, W. H., Buono, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+rates+and+fear+of+phlebotomy+in+pediatric+patients+in+a+genetic+study+of+epilepsy.">Recruitment rates and fear of phlebotomy in pediatric patients in a genetic study of epilepsy.</a> Epilepsy &amp; Behavior. 6, 444-446 (2005).</li><li>Etter, J. F., Neidhart, E., Bertand, S., Malafosse, A., Bertrand, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+saliva+by+mail+for+genetic+and+cotinine+analyses+in+participants+recruited+through+the+internet.">Collecting saliva by mail for genetic and cotinine analyses in participants recruited through the internet.</a> European Journal of Epidemiology. 20, 833-838 (2005).</li><li>Hansen, T. V., Simonsen, M. K., Nielsen, F. C., Hundrup, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection+of+blood,+saliva,+and+buccal+cell+samples+in+a+pilot+study+on+the+danish+nurse+cohort:+Comparison+of+the+response+rate+and+quality+of+genomic+DNA.">Collection of blood, saliva, and buccal cell samples in a pilot study on the danish nurse cohort: Comparison of the response rate and quality of genomic DNA.</a> Cancer Epidemiol Biomarkers Prev. 16, 2072-2076 (2007).</li><li>Van Schie, R. C. A. A., Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva:+A+convenient+source+of+DNA+for+analysis+of+bi-allelic+polymorphisms+of+fc&#947;+receptor+iia+(cd32)+and+fc&#947;+receptor+iiib+(cd16).">Saliva: A convenient source of DNA for analysis of bi-allelic polymorphisms of fc&#947; receptor iia (cd32) and fc&#947; receptor iiib (cd16).</a> Journal of Immunological Methods. 208, 91-101 (1997).</li><li>Dawes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates,+from+salivary+analyses,+of+the+turnover+time+of+oral+mucosal+epithelium+in+humans+and+the+number+of+bacteria+in+an+edentulous+mouth.">Estimates, from salivary analyses, of the turnover time of oral mucosal epithelium in humans and the number of bacteria in an edentulous mouth.</a> Archives of Oral Biology. 48, 329-336 (2003).</li><li>Bahlo, 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=Saliva-derived+DNA+performs+well+in+large-scale,+high-density+single-nucleotide+polymorphism+microarray+studies.">Saliva-derived DNA performs well in large-scale, high-density single-nucleotide polymorphism microarray studies.</a> Cancer Epidemiol Biomarkers Prev. 19, 794-798 (2010).</li><li>Hu, Y., 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=Genotyping+performance+between+saliva+and+blood-derived+genomic+dnas+on+the+dmet+array:+A+comparison.">Genotyping performance between saliva and blood-derived genomic dnas on the dmet array: A comparison.</a> PLoS ONE. 7 (3), e33968 (2012).</li><li>Simmons, T. 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=Increasing+genotype-phenotype+model+determinism:+Application+to+bivariate+reading/language+traits+and+epistatic+interactions+in+language-impaired+families.">Increasing genotype-phenotype model determinism: Application to bivariate reading/language traits and epistatic interactions in language-impaired families.</a> Human Heredity. 70, 232-244 (2010).</li><li>Zeugin, J. A., Hartley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+precipitation+of+DNA.">Ethanol precipitation of DNA.</a> Focus. 7, 1-2 (1985).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+Transform.">Fast and accurate short read alignment with Burrows-Wheeler Transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li><li>McKenna, A., 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+Genome+Analysis+Toolkit:+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data.">The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.</a> Genome Res. 20, 1297-1303 (2010).</li><li>DePristo, 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=A+framework+for+variation+discovery+and+genotyping+using+next-generation+DNA+sequencing+data.">A framework for variation discovery and genotyping using next-generation DNA sequencing data.</a> Nature Genetics. 43, 491-498 (2011).</li></ol>

The authors have nothing to disclose.

Das vorliegende Verfahren ist eine optimierte DNA-Extraktionsprotokoll, die deutlich verbesserte Ausbeute von hochmolekularer DNA im Vergleich zu Standardmethoden, ohne dabei DNA-Qualität. Der entscheidende Schritt mit der größten Wirkung auf den Ertrag die meisten war Schritt 5.2, die eine längere Zentrifugation Schritt während Ethanol-Fällung als jeder veröffentlichten Protokoll hier, überprüft mit einer Ausnahme, die nicht weit verbreitet war 11 umfasst. Keine Änderungen in der DNA-Qualität mit ...

Erzielen hoher Qualität für die menschliche DNA genetische Studien ist wichtig, in der Krankheit Gen Entdeckungsprozess. Blut, obwohl erfordern ein invasives Verfahren und auch teurer als Speichelsammlung wird zum Erzeugen immortalisierter Zelllinien als eine unendliche Quelle von DNA oder iPSCs für funktionelle Studien bevorzugt, und manchmal Blut-DNA wird verwendet, wenn Zelllinien nicht vorhanden sind. , Blutgewinnung erfordert jedoch eine ausgebildete phlebotomist und Blut eine kürzere Halbwertszeit als Speichel 1 hat. DNA aus Speichel ist weniger teuer und leichter zu erhalten, da sie gesammelt und durch die Mail ohne die Notwendigkeit für eine Blutentnahme, wodurch die Versuchsperson Bäder weit über den Einzugsbereich der Krankenhäuser und Laboratorien 2. Studienaufnahme kann verbessert werden, wenn die Probanden die Möglichkeit zu geben eine Speichelprobe statt Blut 3, 4. Können Bedenken hinsichtlich der Quantität und Qualität der DNA aus dem Speichel seine weit verbreitete uns eingeschränkt habenTrotz zahlreicher Studien neueren Studien, die die Eignung der gesamte Speichel, mit einem Durchschnitt von 4,3 x 10 5 Zellen pro Milliliter, zum DNA-Test gegenüber den älteren Tupfern Verfahren, die signifikante Mengen an Speichel 2, 3, 4, 5 nicht erhalten hat e, 6. Während eine bescheidene Literatur existiert, die die Eignung der Gesamtspeichel abgeleiteten DNA für die Genotypisierung Anwendungen wie Microarray-basierte Methoden 8, 9, 10, keine Studien haben untersucht, Next Generation Sequencing (NGS). Das Ziel für die Optimierung dieses ganze Speichel DNA Extraktionsprotokoll war es, Menge und Qualität für Anwendungen in der Genetik einer kosteneffektiven Weise, die leicht in Labore mit gemeinsamen Reagenzien und Verbrauchsmaterialien umgesetzt zu maximieren. DNA-Extraktion aus Speichel erfordert mehrere Verfahren: 1) Sammlung und Lagerung, 2) Zell-Lyse, 3) RNase-Behandlung, 4) Proteinfällung, 5) Ethanol-Fällung, 6) DNA Rehydrierung. Die DNA-Stabilisierungspuffer-Lösung, beschriebenvorher 2, Funktionen angemessen unverändert. Kein Versuch zur Optimierung der RNase-Behandlung und DNA Rehydratisierung Schritte wurde. Für jeden weiteren Schritt wurden mehrere Variablen, die Rendite auswirken könnten identifiziert. Jede Variable wurde einzeln manipuliert und Verbesserung der Ausbeute und Qualität wurde statistisch ausgewertet. Für Variablen, die gezeigt wurden, um die Ausbeute und / oder DNA-Qualität zu verbessern, wurden die optimalen Werte in der letzten Protokoll enthalten.

<table border="0" cellpadding="0" cellspacing="0" width="260">
	<tbody>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">15ml Centrifuge Tubes</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">12-565-268</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Cell Lysis Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158908</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Proteinase K</td>
			<td style="width:88px;">Sigma</td>
			<td style="width:84px;">P6556</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Protein Precipitation Solution</td>
			<td style="width:88px;">Qiagen</td>
			<td style="width:84px;">158912</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Isopropanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">A416-4</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Glycogen</td>
			<td style="width:88px;">EZ-BioResearch</td>
			<td style="width:84px;">S1003</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">70% Ethanol</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">04-355-305</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">Tris-EDTA (TE)</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP2473-1</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">NaCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">AC194090010</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Tris HCl</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP1757-100</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:88px;">EDTA(0.5M) Solution</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">03-500-506</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:88px;">Sodium Dodecyl Sulfate</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">BP166-100&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Name of Equipment</td>
			<td style="width:88px;">Distributor</td>
			<td style="width:84px;">Catalog#</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:88px;">Analog Vortex Mixer</td>
			<td style="width:88px;">Fisher</td>
			<td style="width:84px;">02-215-365</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:88px;">Centrifuge 5810R</td>
			<td style="width:88px;">Eppendorf</td>
			<td style="width:84px;">5811 000.010</td>
		</tr>
	</tbody>
</table>

collection and extraction of saliva dna for next generation sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

Um die optimalen Parameter zu bestimmen für die DNA-Extraktion eine Reihe von gepaarten DNA-Extraktionen durchgeführt wurde. Eine einzelne Speichelprobe wurde aufgeteilt, und jede Portion mit einem von zwei möglichen Werten für eine bestimmte Variable getestet. Mindestens acht Wiederholungen jeder gepaarten Test durchgeführt (beispielsweise wurde ein einzelner Speichelprobe aliquotiert, um die Extraktion mit oder ohne anfängliche 50 ° C Inkubation Test). Gesamt-DNA-Ausbeute, die 260/280 Wert, desto 260/2...

Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

Collection and Extraction of Saliva DNA for Next Generation Sequencing

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Sammlung und Extraktion von DNA-Speichel für Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

collection-and-extraction-of-saliva-dna-for-next-generation-sequencing

Research

JoVE Journal

Medicine

38.8K Aufrufe.  The Research Institute at Nationwide Children's Hospital. DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Serie: Collection and Extraction of Saliva DNA for Next Generation Sequencing

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Erzeugung von Alginat-Mikrokugeln für biomedizinische Anwendungen

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Forschung

Medizin

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

Die α-Test: Rapid Zellfreie CD4-Zählung mit ganzen Speichel

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Sammlung Protokoll zur menschlichen Pankreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

Eine Next-Generation Tissue Microarray (ngTMA) Protokoll zur Biomarker-Studien

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

Integration von Nass- und Trocken Bench Prozesse werden so optimiert Gezielte Next-Generation Sequenzierung von Low-Qualität und Low-Menge Tumorbiopsien

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Wirtschaftliche Extraktion von Kieselalgen-DNA mit einem modifizierten Kit

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Autor im Rampenlicht: Kieselalgen-Tests für forensische Ertrinkungsuntersuchungen 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Extraktion von Diatomeen-DNA aus Wasserproben und Geweben

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

Nicht-invasive Biomarker-Entdeckung für die Tränenproteomik mittels LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Autor im Rampenlicht: Weiterentwicklung der Tränenflüssigkeitsanalyse unter Verwendung eines standardisierten Protokolls für die Proteomikforschung 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Eine Proteinsuspensions-Trapping-Probenvorbereitung für die Tränenproteomik mittels Flüssigkeitschromatographie-Tandem-Massenspektrometrie

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...