This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

وافقت هذه الدراسة من قبل مجلس المراجعة المؤسسية للمستشفى شونام الجامعة الوطنية هواسون وجامعة ولاية كارولينا الجنوبية. وقدمت عينات TMA تتألف من سرطان الثدي ومطابقة الأنسجة الطبيعية للثدي المجاورة من قبل البنك الحيوي من مستشفى شونام الجامعة الوطنية هواسون، وهو عضو في شبكة البنك الحيوي كوريا. 1. الحل تحضير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 1 لتر من الدناز وريبونوكلياز المياه مجانا. جعل جميع الحلول الكيماوية التي تستخدم diethylpyrocarbonate (DEPC) المياه المعالجة. علاج 1 لتر من الماء مع 500 ميكرولتر من DEPC واحتضان لمدة 3 ساعات في درجة حرارة الغرفة. تعقيم المياه إلى تعطيل DEPC. تحذير: لا يستنشق DEPC لأنه من المعروف مسرطنة. </li><li style=";text-align:right;direction:rtl"> إعداد برنامج تلفزيوني 1X من 10X PBS باستخدام المياه DEPC المعالجة. </li><li style=";text-align:right;direction:rtl"> إعداد 20x ومحكمة أمن الدولة (حل 175.3 غرام من كلوريد الصوديوم و 88.2 غرام من سترات الصوديوم في 800 مل من الماء، وضبط درجة الحموضة إلى 7.0 مع انخفاض قليل في 14 N حل حمض الهيدروكلوريك وضبط مستوى الصوت إلى 1 لتر مع واتإيه)، و 4٪ لامتصاص العرق، و 95٪ من الإيثانول و 80٪ من الإيثانول باستخدام المياه DEPC المعالجة. </li><li style=";text-align:right;direction:rtl"> إضافة 8 مل من ثلاثي إيثانول أمين و 1.05 مل من حمض الهيدروكلوريك في 590 مل من المياه المعالجة DEPC وإضافة 1.5 مل من أنهيدريد الخل لأستلة. </li><li style=";text-align:right;direction:rtl"> تجهيز 50 ميكروغرام / ميكرولتر بروتين كاف في المخزن (50 ملي تريس، حمض الهيدروكلوريك، ودرجة الحموضة 8.0 في DEPC المياه المعالجة). </li><li style=";text-align:right;direction:rtl"> إعداد العازلة التهجين يتألف من 500 ميكرولتر من الفورماميد، 250 ميكرولتر من 20X SSC، 100 ميكرولتر من 50X حل دينهارت لو 12.5 ميكرولتر من تي RNA، 2.5 ميكرولتر من الحمض النووي الرنجة الحيوانات المنوية، 30 ميكرولتر من RVC، 0.02 غرام من عرقلة كاشف و 50 ميكرولتر من الماء DEPC. إعداد الطازجة. </li></ol> 2. أقسام نسيج الثدي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع 5 ميكرون أقسام جزءا لا يتجزأ من البارافين الثابتة الفورمالين مع مشراح وتطبيق قسم لشحن شريحة 14. ملاحظة: وقد أعد أنسجة الثدي المستخدمة هنا في وقت سابق من شبكة البنك الحيوي كوريا. </li></ol> 3. الأنسجة التحضير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة البارافين جيئة وذهابام الأنسجة عن طريق غمر الشرائح في 2X جرة Coplin في زيلين الطازجة لمدة 10 دقيقة لكل منهما وترطيب قسم الأنسجة بنسبة 5 حضانات دقيقة في خفض تركيزات الإيثانول (100٪، 95٪ و 80٪) في [ده 2 O. تنفيذ هذه الخطوة في غطاء الدخان. </li><li style=";text-align:right;direction:rtl"> تزج الأنسجة في DEPC المياه المعالجة لمدة 5 دقائق. في هذه المرحلة جعل الحدود حول قسم الأنسجة مع قلم حاجز مسعور. </li><li style=";text-align:right;direction:rtl"> إصلاح الأنسجة مع PFA 4٪ لمدة 15 دقيقة تليها يغسل مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> تزج الأنسجة في 0.3٪ تريتون X-100 / برنامج تلفزيوني لمدة 10 دقيقة تليها يغسل مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> احتضان العينة مع 50 ميكروغرام حل / ميكرولتر بروتين كاف لمدة 15 دقيقة عند 37 درجة مئوية. ثم احتضان أقسام الأنسجة مع ثلاثي إيثانول أمين وأنهيدريد الخل لمدة 5 دقائق في درجة حرارة الغرفة، تليها يغسل مع برنامج تلفزيوني. لا يلزم غسل برنامج تلفزيوني بين العلاج بروتين كاف وثلاثي إيثانول أمين / العلاج أنهيدريد الخليك. </li></ol> 4. التهجين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل الأنسجة بدقة وincuباتي قسم الأنسجة مع العازلة التهجين لمدة 2 إلى 3 ساعات في درجة حرارة الغرفة. عادة 120 العازلة التهجين ميكرولتر ما يكفي لتغطية قسم الأنسجة. </li><li style=";text-align:right;direction:rtl"> تمييع 5&#39;-DIG المسمى LNA التحقيق لتركيز النهائي من 3:00 في 120 حل التهجين ميكرولتر. كما أن تركيز الأسهم عادة ما يكون 40 نانوغرام / ميكرولتر، إضافة 3 ميكرولتر من الأسهم لعازلة تهجين 120 ميكرولتر واحتضان الخليط في درجة حرارة 95 درجة مئوية لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> احتضان قسم الأنسجة مع التحقيق بين عشية وضحاها في 54 درجة مئوية (تيم) في غرفة رطبة. عادة 120 ميكرولتر كافية لتغطية مقطع واحد. إنشاء الرطوبة باستخدام اثنين من مناشف ورقية مبللة. الحجم المثالي للغرفة هو 10 بوصة 8 بوصة. </li><li style=";text-align:right;direction:rtl"> ملاحظة: تيم قد تكون مختلفة لمختلف ميرنا. 54 درجة مئوية هو ذكر لمير 489. </li></ol> 5. صرامة غسل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل الأنسجة مع 5X SSC لمدة 7 دقائق (بعد خطوة التهجين، ريبونوكلياز ظروف خالية لم تعد المطلوبة). </li><li style=";text-align:right;direction:rtl"> غسل قسم الأنسجة خفة دمح 1X SSC لمدة 7 دقائق مرتين في 57 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> غسل قسم الأنسجة مع SSC 0.2x لمدة 7 دقائق عند 57 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> غسل قسم الأنسجة مع SSC 0.2x لمدة 7 دقائق في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> احتضان قسم الأنسجة في برنامج تلفزيوني لمدة 10 دقيقة. </li></ol> 6. حجب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان قسم الأنسجة مع عازلة تمنع في درجة حرارة الغرفة لمدة 1 ساعة في غرفة رطبة (لجعل عازلة تمنع إضافة 2 مل FBS، 10 ميكرولتر من توين-20 و 3 ميكرولتر من جيش صرب البوسنة في 18 م من برنامج تلفزيوني تخزين عازلة تمنع في 4 ° C). </li></ol> 7. الابتدائية الأجسام المضادة الحضانة والتنمية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع 1: 300 المضادة للDIG-AP الأجسام المضادة في عرقلة العازلة واحتضان قسم الأنسجة مع الأجسام المضادة عند 4 درجات مئوية خلال الليل. </li><li style=";text-align:right;direction:rtl"> غسل المقاطع مع غسل العازلة 3 مرات، لمدة 5 دقائق لكل منهما (العازلة التي تقدمها مجموعة تطوير). </li><li style=";text-align:right;direction:rtl"> تطوير قسم الأنسجة مع مجموعة تجارية لالفوسفاتيز القلوية التي تنتج CY-3 ضوء الفلورسنت. </li> &lt;لى&gt; غسل القسم مع غسل العازلة لمدة 5 دقائق. قسم صمة عار مع 100 ميكرولتر من 2.5 نانوغرام / ميكرولتر صبغة هويشت لمدة 5 دقائق تليها غسل 5 دقائق مع غسل العازلة. </li><li style=";text-align:right;direction:rtl"> جبل القسم مع تصاعد عازلة وفقا لبروتوكول الشركة المصنعة. استخدام 15 ميكرولتر في قسم الأنسجة والقضاء حل تصاعد المفرط مع أنسجة مسح. ختم الشريحة مع طلاء الأظافر واضحة لمنع تسرب. </li><li style=";text-align:right;direction:rtl"> مراقبة المجهر قسم الفلورسنت باستخدام مع 10X عدسة الكلي حتى التكبير هو 100X. </li></ol>

<ol>
	<li>Shah, N. R., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+pathogenesis+of+breast+cancer:+Implications+in+diagnosis+and+treatment.">MicroRNAs in pathogenesis of breast cancer: Implications in diagnosis and treatment.</a> World J Clin Oncol. 5, 48-60 (2014).</li><li>Tang, H., 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=miR-185+suppresses+tumor+proliferation+by+directly+targeting+E2F6+and+DNMT1+and+indirectly+upregulating+BRCA1+in+triple-negative+breast+cancer.">miR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.</a> Mol Cancer Ther. 13, 3185-3197 (2014).</li><li>Ye, X. 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=Epigenetic+silencing+of+miR-375+induces+trastuzumab+resistance+in+HER2-positive+breast+cancer+by+targeting+IGF1R.">Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.</a> BMC Cancer. 14, 134 (2014).</li><li>Oneyama, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+downregulation+of+mTOR/FGFR3+controls+tumor+growth+induced+by+Src-related+oncogenic+pathways.">MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.</a> Oncogene. 30, 3489-3501 (2011).</li><li>McGuire, A., Brown, J. A., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+breast+cancer:+the+potential+of+miRNA+for+diagnosis+and+treatment+monitoring.">Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring.</a> Cancer Metastasis Rev. 34, 145-155 (2015).</li><li>Quesne, 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=Biological+and+prognostic+associations+of+miR-205+and+let-7b+in+breast+cancer+revealed+by+in+situ+hybridization+analysis+of+micro-RNA+expression+in+arrays+of+archival+tumour+tissue.">Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue.</a> J Pathol. 227, 306-314 (2012).</li><li>Fernandez, 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=miR-340+inhibits+tumor+cell+proliferation+and+induces+apoptosis+by+targeting+multiple+negative+regulators+of+p27+in+non-small+cell+lung+cancer.">miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.</a> Oncogene. 34, 3240-3250 (2015).</li><li>Yu, L., Zhang, J., Guo, X., Li, Z., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-224+upregulation+and+AKT+activation+synergistically+predict+poor+prognosis+in+patients+with+hepatocellular+carcinoma.">MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma.</a> Cancer Epidemiol. 38, 408-413 (2014).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-146+up-regulation+predicts+the+prognosis+of+non-small+cell+lung+cancer+by+miRNA+in+situ+hybridization.">microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.</a> Exp Mol Pathol. 96, 195-199 (2014).</li><li>Kriegel, A. J., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+in+situ+hybridization+for+formalin+fixed+kidney+tissues.">MicroRNA in situ hybridization for formalin fixed kidney tissues.</a> J Vis Exp. , e50785 (2013).</li><li>Ucar, 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=miR-212+and+miR-132+are+required+for+epithelial+stromal+interactions+necessary+for+mouse+mammary+gland+development.">miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.</a> Nat Genet. 42, 1101-1108 (2010).</li><li>Nuovo, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+microRNAs+in+paraffin+embedded,+formalin+fixed+tissues+and+the+co-localization+of+their+putative+targets.">In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.</a> Methods. 52, 307-315 (2010).</li><li>Kierzek, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+locked+nucleic+acid+residues+on+the+thermodynamic+properties+of+2'-O-methyl+RNA/RNA+heteroduplexes.">The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.</a> Nucleic Acids Res. 33, 5082-5093 (2005).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> CSH Protoc. 2008, (2008).</li><li>Li, 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=Sequential+expression+of+miR-182+and+miR-503+cooperatively+targets+FBXW7,+contributing+to+the+malignant+transformation+of+colon+adenoma+to+adenocarcinoma.">Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma.</a> J Pathol. 234, 488-501 (2014).</li></ol>

There is nothing to disclose.

وكان الهدف من هذه الدراسة هو تحديد مستوى التعبير ميرنا في أنسجة سرطان الثدي باستخدام ميرنا في الموقع التهجين. وتجدر الإشارة إلى أنه في هذه التجربة، وتحسين كل الظروف لأنسجة سرطان الثدي. قد تكون هناك حاجة مزيد من التحسين لأنواع الأنسجة الأخرى. ويجب بذل كل الحلول م?...

سرطان الثدي هو من بين الأورام الخبيثة الأكثر شيوعا التي تؤثر على السكان الإناث في جميع أنحاء العالم. وذكرت أكثر من 1.3 مليون حالة إصابة بسرطان الثدي كل عام في جميع أنحاء العالم 1،2. على الرغم من أن الخلايا السرطانية قد تعتبر تقليديا متجانسة من الناحية البيولوجية والتكاثري للغاية، أصبح من الواضح أن سرطان الثدي هو جينيا وغير متجانسة سريريا. مقاومة للعقاقير المضادة للسرطان انتشارا هي السمة المميزة لسرطان الثدي المتقدمة، مما أدى إلى وفيات في الغالبية العظمى من المرضى من خلال تسهيله من تطور السرطان وبعد ورم خبيث 3. الرنا الميكروية (miRNAs) هي فئة من جزيئات الحمض النووي الريبي الصغيرة حوالي 22 النيوكليوتيدات طويلة أن تنظيم التعبير الجيني عن طريق تثبيط عملية الترجمة مرنا أو التوسط مرنا تدهور 4. وقد تم تحديد أكثر من 2000 miRNAs مختلفة حتى الآن في البشر، التي ترتبط الأمراض التي تصيب الإنسان 5. مجموعة متزايدة من الدراسات هفأظهرت الإلكترونية التي miRNAs يمكن أن تعمل كما المسرطنة والمكثفات الورم وغالبا ما dysregulated في الأورام 6. miRNAs تؤثر بشدة كل من تكون الأورام الأولية عن طريق التحكم في الهيئات التنظيمية الرئيسية للتقدم دورة الخلية، الشيخوخة، موت الخلايا المبرمج والالتهام الذاتي، جنبا إلى جنب مع تلك الحركة الخلايا السرطانية، والغزو والانبثاث 7. ارتبط التعبير شاذ ميرنا أيضا مع الآثار السريرية مثل المرحلة، والتمايز، والتشخيص والاستجابة إلى العلاج المساعد 8. على وجه الخصوص، وزيادة يرتبط ارتباطا مير 146 التعبير مع سوء أحوال الطقس في مرضى سرطان الرئة 9. وقد أثبتت دراسة أخرى أن فقدت التعبير عن ميرنا let7b مرتبط الظواهر الخبيثة، وبالتالي، وضعف البقاء على قيد الحياة 6. فهم أنواع من الخلايا والهياكل التي تعبر عن miRNAs هو جزء مهم من فهم آليات يمكن من خلالها إحداث تغييرات في ميرنا خلية تأثير التعبير والأنسجة الظواهر 10. يتم أخذ بعض miRNAs وجدت أن يفرز في الخلايا اللحمية في الخلايا الظهارية وتعمل خصيصا على أهدافها. في السياق نفسه، تفرز مير 212 و مير-132 التي سدى وتنظيم التفاعلات الظهارية انسجة اللازمة لنمو الأقنية خلال تطوير الغدة الثديية 11. الهدف العام من هذه الورقة هو شرح بروتوكول مفصلة للكشف عن التعبير ميرنا في أنسجة سرطان الثدي من خلال ميرنا في الموقع التهجين. لقد الأمثل جميع الظروف لفحص مستويات التعبير ميرنا-489 في سرطان الثدي عينات المرضى. تحقيقا لهذه الغاية، استخدمنا DIG المسمى مقفل النووي حمض (LNA) التحقيق في تجربتنا. وكان بعض من النيوكليوتيدات لها لتعديل حالات الغدد، وهذا هو، والجسر الذي يربط بين الميثيلين 2 &quot;ذرة أكسجين و 4&quot; ذرة الكربون في العمود الفقري ريبوز الذي يحدد النوكليوتيدات بحيث يبقى &quot;في مكان مغلق&quot; بعد التهجين. ونتيجة لذلك، هو أكثر صعوبةللمجمع المهجنة لتفسد 12،13.

<table><tbody><tr><td>ELF-97</td><td>Life technology&amp;nbsp;</td><td>E6604</td><td></td></tr><tr><td>50x Denhard&#039;t&amp;nbsp;</td><td>Life technology&amp;nbsp;</td><td>750018</td><td></td></tr><tr><td>t-RNA</td><td>Roche</td><td>109541</td><td>Reconstitute in DEPC water</td></tr><tr><td>Herring Sperm DNA</td><td>Promega&amp;nbsp;</td><td>D1815</td><td></td></tr><tr><td>Roche Blocking&amp;nbsp;</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>RVC</td><td>Fisher</td><td>50-812-650</td><td>Before use spin down it at 16.1 RCF and take supernatant&amp;nbsp;</td></tr><tr><td>miR-489 probe&amp;nbsp;</td><td>Exiqon</td><td>38599-01</td><td></td></tr><tr><td>Nuclease free BSA</td><td>Roche&amp;nbsp;</td><td>711454</td><td></td></tr><tr><td>Primary antibody-anti DIG</td><td>Roche&amp;nbsp;</td><td>11093274910</td><td></td></tr><tr><td>Diethyl Pyrocarbonate&amp;nbsp;</td><td>Sigma</td><td>1609478</td><td></td></tr></tbody></table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

تم استخدام أنسجة الأورام السرطانية الثديية البشرية لتحديد التعبير ميرنا-489. تم العثور على الثديية قناة الغدة والخلايا الظهارية للتعبير عن أعلى بكثير ميرنا-489 مستويات من الأنسجة المجاورة ورم في اثنين من المرضى (الشكل 1A و 1B). يدل هذا ...

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

تحليل إكلينيكية من ميرنا التعبير في سرطان الثدي الأنسجة باستخدام ميرنا<em&gt; في الموقع</em&gt; التهجين

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Research

JoVE Journal

Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K Views.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Video: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

تسلسل ميكرورنا أثر رجعي: الحمض النووي التكميلية المكتبة إعداد بروتوكول باستخدام عينات الفورمالين--الثابتة جزءا لا يتجزأ من البارافين الحمض النووي الريبي

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Genetics

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

ميرنا الأنسجة على حدة "التعبير التنميط" في "أقسام القلب الماوس باستخدام" التهجين في الموقع

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

الكشف عن لوحة مخصصة MicroRNA المتداولة في المرضى الذين يعانون من سرطان القولون والمستقيم المنتشر

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Cancer Research

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

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

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

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

الكشف microRNA في أورام البروستات بواسطة PCR الوقت الحقيقي الكمية (qPCR)

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

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

تحليل ميرنا اندماجي علاجات لتنظيم دورة الخلية والأوعية

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

التنميط من MicroRNAs الاستروجين التنظيم في خلايا سرطان الثدي

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

الأمثل للتحليل متعدد التعبير المستندة إلى الجيش الملكي النيبالي استخدام المواد الأرشيفية سرطان الثدي

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

Bioengineering

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

ميرنا تحليلات التعبير في سرطان البروستاتا الأنسجة السريرية

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

الرنا الميكروي<em&gt; في الوضع الطبيعي</em&gt; التهجين للأنسجة الكلى الفورمالين الثابتة

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Biology

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

عزل الرنا غير المكودة الصغيرة من مصل الإنسان

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

تحليل إكلينيكية من ميرنا التعبير في سرطان الثدي الأنسجة باستخدام ميرنا

Summary

Explore More Videos

Chapters in this video