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

הערה: לפני מתן דגימות רוק כל הנושאים נתנו הסכמה מדעת התואמת את הקווים המנחים לטיפול בבני אדם בבית החולים לילדים בפריסה ארצית. .1 רוק איסוף ואחסון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני אוסף רוק, להבטיח כי הפה של הנושא הוא ללא מזון או חומרים זרים אחרים על ידי בעל העניין לשטוף את פיהם במים והימנעות אכילה או שתייה במשך 30 דקות לפני איסוף הדגימה. </li><li style=";text-align:right;direction:rtl"> פתח צינור צנטריפוגות 15 מ&quot;ל עם 2.5 מ&quot;ל של חיץ ייצוב DNA 2 והקפד להימנע מלגעת בחלק הפנימי של הכובע או צינור, ויש לי את הנושא לירוק 2.5 מ&quot;ל של רוק לפתרון החיץ. שים לב: איסוף יותר מ 2.5 מ&quot;ל של רוק יכול להוביל להשפלת מדגם מיחס מספק של מדגם לחיץ ייצוב DNA. איסוף רוק מעט מדי יפחית תשואות הצפויות מהפרוטוקול. כדי להעריך את היקפי גבייה, להשתמש במעברי הצבע הממוספרים בצד של tהוא הצינור. </li><li style=";text-align:right;direction:rtl"> להחליף כובע ומערבבים על ידי היפוך עד לקבלת התערובת הומוגני. טלטול נמרץ הוא לא הכרחי. אחסן את הדגימות ב RT עבור אחסון לטווח קצר או 4 ° C לאחסון לטווח ארוך (&gt; 3 חודשים). </li></ol> 2 הכנות ראשוניות ותמוגה תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני תחילת ההפקה, לחמם אמבט מים ל37 מעלות צלזיוס, ולהכין דלי קרח. שלושה 15 צינורות מ&quot;ל צנטריפוגות חרוטי יהיה צורך עבור כל דגימה שחולצה. שלושה צינורות ישמשו כדי להחזיק את התא גלולה וחלבון, gDNA חילוץ הסופי, וsupernatants isopropanol ואתנול. </li><li style=";text-align:right;direction:rtl"> להשיג דוגמאות קרקע מאחסון, ודגימות הפכו כמה פעמים ואז מערבולת במהירות בינונית במשך 15 שניות. </li><li style=";text-align:right;direction:rtl"> לוותר 2.5 מ&quot;ל של מדגם לתוך צינור צנטריפוגות נקי 15 מ&quot;ל, ולהוסיף 5 מ&quot;ל של תא תמוגה פתרון. מערבבים את המדגם 50 פעמים על ידי היפוך, ולדגור על RT למשך 30 דקות. </li></ol> הסרת .3 RNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 40 μl של RNase סולution ב100 מ&quot;ג / מ&quot;ל, ולדגור על 37 מעלות צלזיוס למשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> הסר את המדגם מאמבט המים 37 מעלות צלזיוס ומגניבה על קרח למשך 3 דקות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר הדגירה RNase, להעלות את הטמפרטורה של המים באמבטיה ל65 מעלות צלזיוס למשך הצעד להחזרת נוזלי DNA של הפרוטוקול. </li></ol></li></ol> .4 חלבון והסרת שומנים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 50 μl של proteinase K פתרון ב20 מ&quot;ג / מ&quot;ל, לערבב כמה פעמים על ידי היפוך, ולדגור על RT למשך תקופה מינימאלית של 30 דקות. הערה: זוהי נקודת השהיה אפשרית לפרוטוקול. לאחר התוספת של proteinase K הפתרון, המדגם יכול להיות מאוחסן על 4 מעלות צלזיוס עד ליכול להסתיים החילוץ. האחסון בC ° 4 עד 24 שעה לא הוצג להיות השפעה משמעותית על תשואות מיצוי או איכות DNA. אחסון לטווח ארוך, בשלב זה לא נבדק. </li><li style=";text-align:right;direction:rtl"> הוספת 1.7 מ&quot;ל של פתרון משקעים חלבון, מערבולת במרץ ל20 שניות במהירות גבוהה, ומניחים על קרח עבור 10 דקות. </li&gt; <li style=";text-align:right;direction:rtl"> ברגע שהדגימות התקררו על קרח עבור 10 דקות, צנטריפוגות במשך 10 דקות ב 3,000 XG ו4 מעלות צלזיוס. החלבונים זירז חייבים ליצור גלולה חזקה להמשיך. אם גלולה היא לא חזקה או הפתרון הוא עדיין מעונן, יכולות להיות מקוררות על דגימות קרח למשך 5 דקות נוספות וצנטריפוגה חוזרת ונשנית. הדגימות חייבות להיות כל הזמן על קרח כדי להבטיח גלולה הדוקה. </li></ol> .5 בידוד וטיהור של gDNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לתוך צינור צנטריפוגות נקי 15 מ&quot;ל, pipet 5 מ&quot;ל של אלכוהול איזופרופיל ו8 μl של טהור גליקוגן פתרון ב20 מ&quot;ג / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> יוצקים את supernatant המכיל gDNA משלב 4.3 לתוך הצינור המכיל האלכוהול איזופרופיל וגליקוגן פתרון, והותיר אחריו את כדור החלבון זירז. ברגע שsupernatant נוסף, לערבב בעדינות המדגם 50 פעמים על ידי היפוך וצנטריפוגות במשך 30 דקות ב 3,000 XG ו4 ° C.. </li><li style=";text-align:right;direction:rtl"> יוצקים את supernatant לאט לתוך צינור 15 מ&quot;ל נקי. לאחר הסרת supernatant, להוסיף 1 מ&quot;ל של אתנול 70% ללשטוף את הכדור על ידי נדנדה לאט ובעדינות נעו אתנול על גלולה זירז כמה פעמים. שמור אתנול בצינור. </li><li style=";text-align:right;direction:rtl"> לאחר השטיפה הראשונית, צנטריפוגות המדגם 1 דקות ב2,000 XG ו20 מעלות צלזיוס. ניתן לעשות צעד צנטריפוגה זה ברמת 4 מעלות צלזיוס או 20 מעלות צלזיוס. אין השפעה משמעותית של טמפרטורה הוכחה בשלב זה. </li><li style=";text-align:right;direction:rtl"> , בעקבות לשטוף וצנטריפוגה הראשוניות של גלולה לשפוך לאט לשטוף אתנול מהצינור וזורקים, ולאחר מכן לבצע לשטוף את השני על ידי חזרה על שלבי 5.3 ו5.4. </li><li style=";text-align:right;direction:rtl"> לאחר הסרת supernatant מלשטוף את השני, לאפשר את גלולה לייבוש באוויר במשך 15 דקות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם המדגם לא התייבש לחלוטין, אוויר יבש לעוד דקות 15. </li></ol></li></ol> .6 Rehydration של gDNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר המדגם התייבש, להוסיף 300 μl של טריס-EDTA לרעננות גלולה gDNA המיובש. </li><li style=";text-align:right;direction:rtl"> מערבולת המדגם במשך 5 שניות במהירות בינונית ומניחים באמבט מים חמים 65 מעלות צלזיוס במשך שעה 1. </li><li style=";text-align:right;direction:rtl"> הסר את הדגימות מאמבט המים ודגירת O / N ב RT. הערה: כל מוצרים וחומרים כימיים המשמשים מפורטים בלוח חומרים, כמו גם לוח 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.

ההליך הנוכחי הוא פרוטוקול מיצוי DNA מותאם שהשתפר במידה ניכרת תשואה של DNA משקל המולקולרי הגבוה בהשוואה לשיטות סטנדרטית, מבלי להתפשר על איכות DNA. השלב הקריטי עם ההשפעה הגדולה ביותר על התשואה ביותר היה צעד 5.2, הכולל צעד צנטריפוגה כבר במהלך משקעים אתנול מכל פרוטוקול שפורסם ...

קבלת DNA באיכות גבוהה עבור מחקרים גנטיים אנושיים היא חיונית בתהליך גילוי גנים למחלות. דם, אם כי דורש הליך פולשני וגם להיות יקר יותר מאשר אוסף רוק, הוא מועדף ליצירת שורות תאים הונצחו כמקור אינסופי של DNA, או iPSCs ללימודים פונקציונליים, ולפעמים DNA הדם משמש בעת שורות תאים אינן זמינות. עם זאת, קבלת דם דורשת phlebotomist מאומן ויש לו דם מחצית חיים קצרים יותר מאשר רוק 1. DNA מרוק הוא פחות יקר וקל יותר להשגה, שכן הוא יכול להיות שנאסף ונשלח באמצעות הדואר ללא הצורך בphlebotomist, ובכך להגדיל בריכות נושא הפוטנציאל הרבה מעבר לאגן ההיקוות של בתי חולים ומעבדות 2. הרשמה למחקר ניתן לשפר את האיכות יש לי נושאים האפשרות של מתן דגימת רוק במקום דם 3, 4. חששות לגבי הכמות והאיכות של DNA מרוק יכולים להיות המוגבלים הנרחבדואר למרות מחקרים שנעשה לאחרונה מחקרים רבים המראים את התאמתו של כל רוק, עם ממוצע של 4.3 x 10 5 תאים למיליליטר, לבדיקות DNA על פני שיטות מטליות buccal המבוגרות שלא לקבל כמויות משמעותיות של רוק 2, 3, 4, 5, 6. בעוד ספרות צנועה קיימת מראה את ההתאמה של DNA רוק השלם נגזר עבור יישומי גנוטיפ כולל שיטות המבוסס על microarray 8, 9, רצף 10, אין מחקרים בחנו הדור הבא (NGS). המטרה לייעול פרוטוקול חילוץ זה כל DNA הרוק הייתה למקסם את הכמות ואיכות עבור יישומי גנטיקה בדרך יעילה עלות שמיושמת בקלות במעבדות עם חומרים כימיים וחומרים נפוצים. מיצוי DNA מרוק דורש מספר הליכים: 1) איסוף ואחסון, טיפול RNase 2) תמוגה תא, 3), 4) ממטרים חלבון, 5) משקעים אתנול, 6) להחזרת נוזלי DNA. פתרון DNA ייצוב הצפת, שתוארבעבר 2, פונקציות כראוי ללא שינוי. שום ניסיון לייעל את טיפול RNase וצעדים להחזרת נוזלי DNA נעשה. לכל שלב שנותר, מספר משתנה שיכול להשפיע על תשואה זוהו. כל אחד ממשתנים היו מניפולציות באופן אישי ושיפור בתפוקה ואיכות הוערך סטטיסטי. עבור משתנים שהוצגו כדי לשפר את התשואה ו / או איכות DNA, הערכים אופטימליים נכללו בפרוטוקול הסופי.

<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.

כדי לקבוע פרמטרים אופטימליים להפקה DNA סדרה של עקירות DNA מזווגות בוצעה. דגימת רוק אחת פוצלה וכל מנה שנבדקה עם אחד משני ערכים אפשריים עבור משתנה נתון. לפחות שמונה חזרות של כל בדיקה לזווג בוצעו (למשל, דגימת רוק אחת הייתה aliquoted לבדוק חילוץ עם או בלי הדגירה C ° 50 ראשונית)....

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.

איסוף והפקת DNA רוק לסידור הדור הבא

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

38.5K Views.  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.

Video: 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.

דור של אלגינט microspheres עבור יישומים ביו

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

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.

Α-מבחן: ראפיד נייד ללא ספירת CD4 באמצעות הרוק שלם

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.

אוסף פרוטוקול לבלב אנוש

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.

פרוטוקול Microarray רקמות הדור הבא (ngTMA) לביומרקר לימודים

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.

אינטגרציה של Wet ותהליכי ספסל יבשים מבצעת אופטימיזציה ממוקד רצף הדור הבא של איכות נמוכה וביופסיות גידול נמוכה כמות

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

מיצוי DNA דיאטומי חסכוני באמצעות ערכה מותאמת

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.

מחבר זרקור: בדיקות דיאטום לבדיקת טביעה משפטית 

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

מיצוי DNA דיאטום מדגימות מים ורקמות

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

גילוי סמנים ביולוגיים לא פולשניים לפרוטאומיקה של דמעות באמצעות 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.

מחבר זרקור: קידום ניתוח נוזל הדמעות באמצעות פרוטוקול סטנדרטי לחקר פרוטאומיקה 

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.

הכנת דגימה ללכידת תרחיף חלבונים עבור פרוטאומיקה של דמעות על ידי כרומטוגרפיה נוזלית-ספקטרומטריית מסה טנדם

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