وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح F32 NS078958 (DTB) وR01 AG033620 (BG).

1. تجميع تستر الطيران <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> آمنة اسطوانة رحلة الى الدائري قف 1 باستخدام المشابك السلسلة. (اترك حوالي 3 سم أسفل الاسطوانة لوزن الطبق.) (ملاحظة: اسطوانة طيران الذي نستعمله هو 90 سنتيمترا وقطره 13.5 سم). </li><li style=";text-align:right;direction:rtl"> إدراج وزن الطبق مع طبقة رقيقة من الزيت المعدني تحت اسطوانة الطيران. </li><li style=";text-align:right;direction:rtl"> قمع آمنة لحزام حامل 2 باستخدام المشبك الدائري والمشبك مخلب. ضبط ارتفاع القمع بحيث الجزء السفلي من القمع هو مطاردة مع الجزء العلوي من الاسطوانة الطيران. (ملاحظة: يجب أن يكون قطرها غيض من قمع أقل من القطر الخارجي للقارورة وضعت في أنابيب قطرة بحيث قارورة لا تقع من خلال.) </li><li style=";text-align:right;direction:rtl"> إدراج قطرة أنبوب في الجزء العلوي من القمع وتأمين استخدام المشبك مخلب. (ملاحظة: نحن نستخدم أنبوب قطرة هذا هو طوله 25 سم اسقاط قارورة من هذا الارتفاع يسمح طرد ثابت من كل الذباب مع قوة موحدة تحتوي على ذبابة يجب أن يكون القطر الداخلي للأنبوب قطرة سليغ.أكبر TLY من القطر الخارجي للقارورة للسماح القارورة لإسقاط بحرية.) </li><li style=";text-align:right;direction:rtl"> قطع ورقة بولكرلميد (ق) إلى الحجم المناسب. (ملاحظة: لمساعدة في إدراج وإزالة ورقة، ويجب أن يكون عرض أصغر قليلا من محيط الداخلي للاسطوانة الطيران). </li><li style=";text-align:right;direction:rtl"> تطبيق طبقة رقيقة من التشابك-فخ إلى ورقة. السماح للجلوس لمدة 1 ساعة قبل الاستخدام. (ملاحظة: اترك مساحة كافية في الجزء العلوي والسفلي من ورقة (حوالي 3 سم) غير المصقول لفهم ورقة عن الإدراج / إزالة.) </li><li style=";text-align:right;direction:rtl"> إدراج ورقة بولي أكريلاميد في الاسطوانة الطيران. </li><li style=";text-align:right;direction:rtl"> تجميع المسار الكاميرا باستخدام الأقواس دعم الصنوبر. (ملاحظة: تأكد من أن الجزء السفلي من المسار يمكن أن تدعم الكاميرا دون عرقلة عدسة ارجع إلى الشكل 1B.) </li><li style=";text-align:right;direction:rtl"> إضافة سدادات والمسمار في مكانه. (ملاحظة: مكان سدادات في المواقع التي تسمح الكاميرا لعرض ورقة من البلاستيك بالكامل في وضع بانورامية.) </li></ol> 2. تشغيل التجربة <ol style=";text-align:right;direction:rtl"> &lt;لى&gt; جمع قارورة من الذباب لفحصها. للحصول على أفضل النتائج، استخدم ما لا يزيد عن 20 الذباب / القارورة. </li><li style=";text-align:right;direction:rtl"> اضغط برفق الذباب إلى أسفل القارورة، افصل، ثم تضاف إلى انخفاض أنبوب والافراج القارورة. (ملاحظة: قارورة تسقط أنبوب قطرة حتى يضرب افتتاح قمع الضيقة عندما يضرب القارورة القمع، وطرد الذباب في الاسطوانة الرحلة.) </li><li style=";text-align:right;direction:rtl"> رفع أنبوب قطرة لإزالة قارورة فارغة. (ملاحظة: قوارير متعددة من الذباب من نفس المجموعة الاختبار يمكن أن يعاير على ورقة واحدة بولكرلميد نجد أن ما يصل إلى 200 الذباب (10 قارورة من الذباب 20 لكل منهما) ويمكن اختبارها وتصويرها بسهولة على ورقة واحدة. </li><li style=";text-align:right;direction:rtl"> إزالة ورقة من البلاستيك ووضعه على سطح مستو أبيض. (ملاحظة: يمكن استخدام لوحة الملصق الأبيض إذا قمم مقاعد البدلاء هي داكنة اللون.) </li><li style=";text-align:right;direction:rtl"> تجميع المسار الكاميرا على ورقة من البلاستيك. يجب أن تكون الكاميرا عالية بما فيه الكفاية فوق ورقة لديك كل من أعلى وأسفل الورقة في مجال الرؤية. </li><li style=";text-align:right;direction:rtl"> حرك الكاميرا على طول الالبريد المسار أثناء الضغط على زر &quot;التقاط&quot; للحصول على صورة بانورامية. </li><li style=";text-align:right;direction:rtl"> قد تحسب عدد الذباب الهبوط في النفط يدويا لكل المحاكمة. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2،2-2،7 لجميع الظروف في تجربة معينة. يمكن إزالة الذباب من ورقة بين كل محاكمة. بدلا من ذلك، يمكن استخدام عدة أوراق، مع ورقة جديدة لكل المحاكمة. </li></ol> 3. جمع البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملفات الصور المفتوحة باستخدام برنامج ImageJ. </li><li style=";text-align:right;direction:rtl"> الصور المحاصيل إذا لزم الأمر ليشمل فقط مساحة الهبوط. (وهذا هو المجال المغلفة في تشابك-فخ.) </li><li style=";text-align:right;direction:rtl"> تحويل الصور إلى 8 بت الرمادي. </li><li style=";text-align:right;direction:rtl"> إنشاء &quot;عتبة&quot; لتصفية خلفية بيضاء. (صورة → → ضبط عتبة). </li><li style=";text-align:right;direction:rtl"> تعيين المعلمات لتعريف كل الطاير باستخدام &quot;تحليل الجزيئات&quot; القائمة. (تحليل → تحليل الجزيئات) تحديد المعايير المستخدمة لتحديد الجسيمات. مع تعيين لدينا ما يصل، نجد أن استخدامسوف مجال 5-90 بكسل 2 ودائرية من 0،4-1،0 تحديد بدقة جميع العينات. </li><li style=";text-align:right;direction:rtl"> قياس موقع كل الطاير باستخدام قائمة ولدت من الإحداثيات لكل الجسيمات. الإحداثي س بالبكسل يمكن تحويلها إلى سم لحساب ارتفاع الهبوط. </li><li style=";text-align:right;direction:rtl"> استيراد الجدول إلى جدول بيانات (مثل Microsoft Excel). </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., 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=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

والكتاب ليس لديهم تضارب المصالح في الكشف عنها.

باستخدام أساليب وصفها هنا، كنا قادرين على تقييم سريع لعدد كبير من المسوخ ذبابة الفاكهة أداء الطيران، وتوفير كفاءة أكبر مما كان ممكنا في السابق. لتجاربنا، ونحن بشكل روتيني فصل الذكور والإناث ورفع لهم في منخفض الكثافة (أقل من 20 الذباب / القارورة) للحد من العدوان ال...

منذ فترة طويلة تستخدم ذبابة الفاكهة لدراسة الأساس الجيني للسلوك 1، وابتكر الباحثون عددا من الطرق لتحليل أنواع مختلفة من السلوك 2-6. كان الذباب مفيدة بشكل خاص في تقديم نماذج مفيدة من الاضطرابات العصبية والعضلية 7. A مقايسة شيوعا التي تستخدم لدراسة السلوك الحركي هو أداء الطيران. اختبار طيران الأصلي هو مفيد لتحديد المسوخ معيبة الطيران وللتقييم الكمي للقدرة طيران 1، لكنه لا يملك العديد من أوجه القصور التي تحد من تطبيقه للشاشات إنتاجية عالية: استخدام اسطوانات المغلفة للنفط هو فوضوي ومرهقة، وميزات معينة مثل طول الاسطوانة وإدخال الذباب في أنبوب مع قوة متغير تقليل الدقة الكمية، وأنه من الصعب استعادة الذباب على الهواء مباشرة من اختبار. للتغلب على هذه القيود، قمنا بتعديل اختبار طيران لتشمل عددا من التحسينات. واضاف نحن &quot;قطرة توأن &quot;لإدخال الذباب للقضاء على التباين بين التجارب والمستخدمين. نحن نستخدم أوراق الأكريليك القابلة للإزالة المغلفة بمادة لاصقة تسمح لتسهيل تنظيف والانتعاش من الذباب الفردية. لقد زاد طول أنبوب رحلة إلى تحسين دقة وموثوقية الكمي. أخيرا ، ونحن نستخدم كاميرا رقمية وبرامج التصوير لحساب ارتفاعات الهبوط من الذباب، ونحن نعتقد أن هذه التحسينات أن تكون مفيدة لأي مختبر مهتمة في إجراء شاشات جينية واسعة النطاق لعيوب في أداء الطيران.

<table border="0" cellpadding="0" cellspacing="0" width="704">
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	<tbody>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Putty knife</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">630147</td>
			<td style="width:185px;"><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Pine back band moulding ( x2 )</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">156469</td>
			<td><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Furring Strip Board</td>
			<td style="width:171px;">Home Depot</td>
			<td style="width:132px;">164704</td>
			<td><a href="http://www.homedepot.com">www.homedepot.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Tangle Trap Insect Trap Coating</td>
			<td style="width:171px;">BioControl Network</td>
			<td style="width:132px;">268941</td>
			<td><a href="http://www.biconet.com">www.biconet.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Laptop Computer </td>
			<td style="width:171px;">Apple</td>
			<td style="width:132px;">_</td>
			<td><a href="http://www.apple.com/mac/">www.apple.com/mac/</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">mineral oil</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">BP26291</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">white poster board</td>
			<td style="width:171px;">Staples</td>
			<td style="width:132px;">247403</td>
			<td><a href="http://www.staples.com">www.staples.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polystyrene weighing dish</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S67091A</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ImageJ Software</td>
			<td style="width:171px;">National Institutes of Health</td>
			<td style="width:132px;">_</td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">digital camera</td>
			<td style="width:171px;">Sony</td>
			<td style="width:132px;">DSC-TX7</td>
			<td style="width:185px;"><a href="http://www.store.sony.com">www.store.sony.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">fine forceps</td>
			<td style="width:171px;">Fine Science Tools</td>
			<td style="width:132px;">_</td>
			<td><a href="http://www.finescience.com">www.finescience.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polycarbonate cylinder (drop tube)</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">8585K62</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">flight cylinder (acrylic)</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">8486K943</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">polycarbonate sheets</td>
			<td style="width:171px;">mcMaster-Carr</td>
			<td style="width:132px;">85585K25</td>
			<td><a href="http://www.mcmaster.com">www.mcmaster.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ring stand (x2)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S47808</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">ring support</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">S47791</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">Three-prong extension clamps (x2)</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">05-769-7Q</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">funnel</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:132px;">10-500-3</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">chain clamps (x2)</td>
			<td style="width:171px;">VWR</td>
			<td style="width:132px;">21573-275</td>
			<td><a href="http://www.vwr.com">www.vwr.com</a></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:216px;">glass vials</td>
			<td style="width:171px;">VWR</td>
			<td style="width:132px;">66020-198</td>
			<td><a href="http://www.vwr.com/">www.vwr.com</a></td>
		</tr>
	</tbody>
</table>

an improved method for accurate and rapid measurement of flight performance in drosophila

وقد ثبت ذبابة الفاكهة ليكون نظام نموذجا مفيدا لتحليل السلوك، بما في ذلك الرحلات الجوية. الأولي اختبار طيران تشارك إسقاط الذباب إلى تخرج المغلفة النفط أسطوانات، شريطة ارتفاع الهبوط مقياسا للأداء الرحلة من خلال تقييم مدى الذباب بعيدا ستسقط قبل إنتاج ما يكفي من التوجه لاجراء اتصالات مع جدار الاسطوانة. نحن هنا تصف إصدار محدث من اختبار الطيران مع أربعة تحسينات كبيرة. الأول، وأضاف لدينا &quot;قطرة أنبوب&quot; لضمان أن جميع الذباب أدخل اسطوانة طيران في سرعة مماثلة بين التجارب، والقضاء على التباين بين المستخدمين. الثانية، ونحن استبدال النفط الطلاء مع الأغطية البلاستيكية القابلة للإزالة المغلفة في تشابك-فخ، لاصقة مصممة لالتقاط الحشرات الحية. الثالثة، ونحن نستخدم اسطوانة أطول لتمكين التمييز أكثر دقة من قدرة الطيران. الرابع نستخدم الكاميرا الرقمية وبرامج التصوير لأتمتة التهديف من أداء الطيران. هذه التحسينات تسمح للالرابالهوية، والتقييم الكمي للسلوك طيران، مفيدة لمجموعات البيانات الكبيرة وشاشات جينية واسعة النطاق.

<html> ويبين الشكل 1A التخطيطي لتجميع اختبار طيران تحديثها. يوضح الشكل 1B تصميم المسار تسمح الكاميرا لالتقاط صورة بانورامية دون عرقلة مجال الرؤية. وتظهر نتائج ممثلة في الشكل 2، حيث من الذباب متحولة الكسول، التي لديها الطيران المعروفة عيب <s...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

نحن هنا تصف طريقة لقياس سريع ودقيق للأداء الطيران في ذبابة الفاكهة، مما يتيح الفرز الفائق الإنتاجية.

لتحسين طريقة لقياس دقة وسرعة الأداء في الطيران<em&gt; ذبابة الفاكهة</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

11.7K Views.  University of Wisconsin-Madison.  نحن هنا تصف طريقة لقياس سريع ودقيق للأداء الطيران في ذبابة الفاكهة، مما يتيح الفرز الفائق الإنتاجية. 

Video: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : طريقة بسيطة لالتنميط الجيني دقيقة

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

عالية الدقة قياس سلوك رائحة يحركها يرقات ذبابة الفاكهة في

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

: نظام الحبل المغناطيسي بالتحقيق البصرية والشمية التحكم الطيران توسط في ذبابة الفاكهة

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

تتبع الرائحة بصريا وساطة خلال الطيران في ذبابة الفاكهة

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

طريقة محسنة لعزل الحمض النووي الريبي من الصنوبر Loblolly ( P. taeda L.) والأنواع الأخرى الصنوبرية

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

التحمل بروتوكول التدريب والمقايسات الأداء الطولية لذبابة الفاكهة الدروسوفيلا

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

قياس عمر في<em&gt; ذبابة الفاكهة الدروسوفيلا</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

طريقة مبتكرة لExosome الكمي وقياس الحجم

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

قياس معدل الأيض في<em&gt; ذبابة الفاكهة</em&gt; باستخدام قياس التنفس

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

عزل الترشيح من الأحماض النووية: وبسيطة وسريعة استخراج الحمض النووي الطريقة

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...