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.

מחקר זה אושר על ידי דירקטוריון הסקירה המוסדית של בית החולים האוניברסיטאי הלאומי Chonnam הוואסון ואונ&#39; הדרום קרוליינה. דגימות TMA מורכבות סרטן שד רקמות שד נורמלים סמוכות מתאימות נמסרו על ידי Biobank של חולי הוואסון אונ&#39; הלאומיים Chonnam, חבר ברשת Biobank קוריאה. 1. פתרון כנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כן 1 ליטר מים חינם DNase ו RNase. הפוך את כל תמיסות כימיות באמצעות diethylpyrocarbonate (DEPC) מים מטופלים. פנקו 1 ליטר מים עם 500 μl של DEPC דגירה במשך 3 שעות בטמפרטורת החדר. חיטוי המים כדי להשבית DEPC. זהירות: אין לשאוף DEPC מאחר וידוע מסרטן. </li><li style=";text-align:right;direction:rtl"> הכן PBS 1x מ 10x PBS באמצעות מים מטופלים DEPC. </li><li style=";text-align:right;direction:rtl"> הכן 20x SSC (לפזר 175.3 גרם של NaCl ו 88.2 גרם של נתרן ציטראט ב 800 מ&quot;ל מים, להתאים את ה- pH ל 7.0 עם ירידה כמה 14 N פתרון של HCl ולהתאים את עוצמת הקול עד 1 ליטר עם וואטאה), 4% paraformaldehyde, 95% אתנול, 80% אתנול באמצעות מים מטופלים DEPC. </li><li style=";text-align:right;direction:rtl"> הוסף 8 מ&quot;ל של triethanolamine ו -1.05 מ&quot;ל של HCl ב 590 מ&quot;ל מים DEPC שטופלו ולהוסיף 1.5 מ&quot;ל של אנהידריד אצטית עבור acetylation. </li><li style=";text-align:right;direction:rtl"> הכן 50 מיקרוגרם / μl proteinase K במאגר (50 mM Tris-HCl, pH 8.0 ב DEPC מים מטופלים). </li><li style=";text-align:right;direction:rtl"> הכן חיץ הכלאה מורכב 500 μl של לפוראמיד, 250 μl של 20x SSC, 100 μl של 50x פתרון של Denhardt, 12.5 μl של t-RNA, 2.5 μl של ה- DNA זרע הרינג, 30 μl של RVC, 0.02 גרם של מגיב חסימת 50 μl מי DEPC. כן טרי. </li></ol> 2. סעיפי רקמות שד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכי 5 מיקרומטר קבוע פורמלין חלקי פרפין מוטבע עם microtome ולהחיל את הסעיף לחייב שקופית 14. הערה: רקמת השד משמש כאן הוכנה בעבר מרשת Biobank קוריאה. </li></ol> 3. רקמות הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר פרפין הלוך ושובמ הרקמה ידי טבילה שקופיות לתוך 2x צנצנת Coplin קסילן טרי במשך 10 דקות כל אחד ו רעננותם קטע רקמה על ידי 5 incubations דקות בהפחתת ריכוזי אתנול (100%, 95% ו -80%) ב DDH 2 O. בצע פעולה זו במנדף. </li><li style=";text-align:right;direction:rtl"> לטבול את רקמת DEPC מי מטופלים במשך 5 דקות. בשלב זה יש לבצע גבולות סביב קטע רקמה עם עט מחסום הידרופובי. </li><li style=";text-align:right;direction:rtl"> לתקן את הרקמה עם 4% PFA במשך 15 דקות ואחריו לשטוף עם PBS. </li><li style=";text-align:right;direction:rtl"> לטבול את הרקמה ב 0.3% Triton X-100 / PBS במשך 10 דקות ואחריו לשטוף עם PBS. </li><li style=";text-align:right;direction:rtl"> דגירה המדגם עם 50 מיקרוגרם / פתרון μl proteinase K עבור 15 דקות ב 37 מעלות צלזיוס. ואז דגירה סעיפים רקמה עם triethanolamine ו אנהידריד אצטית עבור 5 דקות בטמפרטורת החדר, ואחריו לשטוף עם PBS. אין לשטוף PBS נדרשת בין טיפול proteinase K ו triethanolamine / טיפול אנהידריד אצטית. </li></ol> הכלאה 4. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו את הרקמה ביסודיות incuבייט בסעיף רקמות עם חיץ הכלאה ל -2 עד 3 שעות בטמפרטורת החדר. בדרך כלל 120 חיץ הכלאה μl הוא מספיק כדי לכסות את קטע רקמה. </li><li style=";text-align:right;direction:rtl"> לדלל 5&#39;-DIG שכותרתו בדיקה LNA כדי הריכוז הסופי של 3 PM ב 120 פתרון הכלאה μl. ככל ריכוז המניות בדרך כלל הוא 40 ng / μl, להוסיף 3 μl של המניה ל -120 μl חיץ הכלאה ו דגירה התערובת על 95 מעלות צלזיוס למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> קטע רקמת דגירה עם הלילה חללי על 54 מעלות צלזיוס (Tm) בתא לח. בדרך כלל 120 μl הוא מספיק כדי לכסות חלק אחד. צור לחות באמצעות שתי מגבות נייר רטובות. הגודל האידיאלי של החדר הוא 10 אינץ &#39;על 8 אינץ&#39;. </li><li style=";text-align:right;direction:rtl"> הערה:&#39;אני עשוי להיות שונה עבור מירנה שונה. 54 ° C הוא אזכורים עבור miR-489. </li></ol> 5. חומרא Wash <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטוף את הרקמה עם 5x SSC עבור 7 דקות (לאחר שלב הכלאה, תנאים חינם RNase אינם נחוצים עוד?). </li><li style=";text-align:right;direction:rtl"> שטפו את השנינות קטע רקמהh 1x SSC עבור 7 דקות פעמיים ב 57 ° C. </li><li style=";text-align:right;direction:rtl"> שטפו את קטע רקמה עם SSC 0.2X עבור 7 דקות ב 57 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> שטפו את קטע רקמה עם SSC 0.2X עבור 7 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> קטע רקמה מדגירים PBS במשך 10 דקות. </li></ol> 6. חסימה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה קטע רקמה עם חסימת חיץ בטמפרטורת החדר למשך שעה 1 בתא לח (כדי להפוך חיץ חסימת להוסיף 2 FBS מ&quot;ל, 10 μl של Tween-20 ו -3 μl של BSA ב -18 מ &#39;של PBS. חסימת חנות חיץ ב 4 מעלות צלזיוס). </li></ol> 7. דגירה נוגדן ראשוני ופיתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל 1: 300 נוגדן אנטי DIG-AP בחסימת מאגר דגירת קטע הרקמה עם נוגדן ב 4 מעלות צלזיוס למשך הלילה. </li><li style=";text-align:right;direction:rtl"> שטוף את החלקים עם חיץ לשטוף 3 פעמים, במשך 5 דקות כל אחד (חיץ שמספק ערכת פיתוח). </li><li style=";text-align:right;direction:rtl"> לפתח את קטע הרקמה עם ערכה מסחרית עבור phosphatase אלקליין שמייצר אור פלורסנט CY-3. </li> &lt;li&gt; שטף את החלק עם חיץ לשטוף למשך 5 דקות. סעיף הכתם עם 100 μl של 2.5 ng / לצבוע Hoechst μl במשך 5 דקות ואחריו לשטוף 5 דקות עם חיץ לשטוף. </li><li style=";text-align:right;direction:rtl"> הר הקטע עם הרכבת חיץ על פי הפרוטוקול של היצרן. השתמש 15 μl לכל קטע רקמה ולנגב פתרון הרכבה מוגזם עם רקמת לנגב. חותם את השקופית עם לק ברור למנוע דולף. </li><li style=";text-align:right;direction:rtl"> שימו מיקרוסקופ פלורסנט סעיף משתמש עם 10X עדשה כך שסך כל ההגדלה היא 100X. </li></ol>

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There is nothing to disclose.

מטרת המחקר הנוכחי הייתה לקבוע את רמת הביטוי מירנה ברקמות סרטן השד באמצעות מירנה הכלאה באתרה. יש לציין כי בניסוי זה, כל התנאים היו אופטימיזציה עבור רקמת סרטן השד. אופטימיזציה נוספת עשויה להידרש סוגי רקמות אחרות. כל הפתרונות חייב להיעשות עם מים DEPC וכל מכולות לשמש י...

סרטן השד הוא בין סוגי הסרטן הנפוצים ביותר המשפיעים על האוכלוסייה הנשית על פני הגלובוס. יותר מ -1.3 מיליון מקרים של סרטן השד מדווחים מדי שנה ברחבי עולם 1,2. למרות תאים סרטניים נחשבו באופן מסורתי כמו ביולוגית הומוגנית מאוד שגשוג, זה הפך להיות ברור כי סרטן השד הוא גנטי קליני הטרוגנית. עמידות לתרופות אנטי סרטניות רווחות היא סימן היכר של סרטן השד מתקדם, שמובילה את התמותה ברוב המכריע של מטופלים באמצעות הסיוע של גרורות מרחק התקדמות וסרטן 3. מיקרו RNA (miRNAs) הם קבוצה של מולקולות RNA קטנות כ 22 נוקליאוטידים המווסתים ביטוי גנים על ידי חסימת תרגום mRNA או בתיווך mRNA השפלה 4. יותר מ -2,000 miRNAs השונה עד כה זוהה בבני אדם, אשר מקושרים מחל אנושיות 5. מערך הולך וגדל של מחקרים havהדואר הוכיח כי miRNAs יכול לתפקד כמו מוטציות מדכאי גידול ובדר&quot;כ dysregulated בגידולים 6. miRNAs מאוד להשפיע כל tumorigenesis העיקרי על ידי שליטה על הרגולטורים העיקריים של התקדמות מחזור התא, הזדקנות, אפופטוזיס autophagy, יחד עם אלה של תנועתיות תאים סרטניים, פלישה גרורה 7. ביטוי Aberrant מירנה נקשר גם עם השלכות קליניות כגון במה, בידול, הפרוגנוזה ואת תגובה לטיפול אדג&#39;ובנטי 8. בפרט, גדל ביטוי miR-146 הוא מתואם עם פרוגנוזה גרועה בחולי סרטן ריאות 9. מחקר אחר הראה כי ביטוי אבוד של מירנה-let7b קשור פנוטיפים ממאירים, וכתוצאה מכך, 6 הישרדות עניה. הבנת הסוגים של תאים ומבנים מבטאים miRNAs היא חלק חשוב להבין את המכניקה שדרכו שינויים בתא השפעת ביטוי מירנהרקמת פנוטיפים 10. miRNAs מסוים מצא להיות מופרשים בתאי סטרומה נלקח על ידי תאי אפיתל ולפעול עליהם במיוחד את המטרות שלהם. באותה רוח, miR-212 ומיר-132 מופרשים על ידי stroma ולהסדיר את אינטראקציות סטרומה אפיתל הנדרשים תולדה ductal במהלך התפתחות בלוטת החלב 11. המטרה הכללית של מאמר זה היא להסביר פרוטוקול מפורט לזהות ביטוי מירנה ברקמות סרטן השד באמצעות מירנה הכלאה באתרה. יש לנו אופטימיזציה כל התנאים כדי assay את רמות ביטוי מירנה-489 בדגימות חולות סרטן השד. לשם כך, השתמשנו DIG שכותרתו נעול חומצות גרעין (LNA) בדיקה בניסוי שלנו. חלק נוקלאוטידים המקורי היה שינוי LNA, כלומר, גשר מתילן חיבור &#39;אטום חמצן 4&#39; 2 אטום פחמן של עמוד השדרה ריבוז שמתקן נוקלאוטיד כזה שהוא נשאר &quot;נעול במקום&quot; לאחר כלאה. כתוצאה מכך, זה הרבה יותר קשהעבור מתחם ההכלאה לפגל 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.

רקמת סרטן שד אדם שמשה כדי לקבוע ביטוי מירנה-489. צינור בלוטת חלב ותאי אפיתל נמצאו להביע מירנה-489 גבוהות משמעותי רמות מאשר רקמת גידול סמוכה בשני חולים (איור 1A ו -1 B). זה מראה בבירור כי ביטוי מירנה-489 אבד רקמת גידול, דבר המצביע על פעילות מד...

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.

ניתוח Clinicopathological ביטוי מירנה שד רקמות סרטן על ידי שימוש מירנה<em&gt; באתרו</em&gt; כלאה

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.

רצף MicroRNA רטרוספקטיבה: DNA משלימים ספריית הכנת פרוטוקול באמצעות פורמלין-קבוע RNA פרפין-מוטבע דגימות

&#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.

MiRNA רקמות ספציפיות ביטוי פרופיל בהלב העכבר באמצעות סעיפים באתרו של הכלאה

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.

זיהוי של לוח מותאם אישית MicroRNA במחזור בחולים עם סרטן המעי הגס גרורתי

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.

MicroRNA איתור גידולים הערמונית ב כמותי בזמן אמת PCR (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.

ניתוח של miRNA קומבינטורית טיפולים כדי להסדיר מחזור התא, אנגיוגנזה

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.

פרופיל של מיקרו RNA המוסדר אסטרוגן בתאי סרטן השד

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.

אופטימיזציה של וזמינותו הביטוי מבוסס-RNA מולטיפלקס באמצעות חומר ארכיוני של סרטן השד

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).

ניתוח ביטוי מירנה ברקמות קליניות בסרטן הערמונית

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.

MicroRNA<em&gt; באתרו</em&gt; הכלאה לרקמות כליה פורמלין קבועה

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.

בידוד של קטן noncoding RNAs מסרום האדם

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 ...

ניתוח Clinicopathological ביטוי מירנה שד רקמות סרטן על ידי שימוש מירנה

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