Name: microrna detection in prostate tumors by quantitative real-time pcr (qpcr)
Uploaded: 2012-05-16T12:00:00
Description: Watch this Scientific Journal Video about MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR at JoVE.com

وقد تم تمويل هذا العمل من قبل المعهد الكندي لابحاث السرطان في المجتمع، لا تمنح. 019038.

<html> 1. البروستاتا جمع العينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع عينات من البروستاتا في وقت استئصال البروستاتا. توجه العينة باستخدام معالم التشريحية. ورسمت على البروستاتا والحويصلات المنوية على النحو التالي: حق أخضر الجانب، جانب اليسار الزرقاء. </li>...

<ol>
	<li>Reinhart, B. J. <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+plants.">MicroRNAs in plants.</a> Genes Dev. 16, 1616-1616 (2002).</li><li>Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+microRNA+Mir-14+suppresses+cell+death+and+is+required+for+normal+fat+metabolism.">The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism.</a> Curr. Biol. 13, 790 (2003).</li><li>Bueno, M. J., Perez, d. e. C. a. s. t. r. o., I, ., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation+pathways+by+microRNAs.">Control of cell proliferation pathways by microRNAs.</a> Cell Cycle. 7, 3143-3143 (2008).</li><li>Friedman, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92 (2009).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-281 (2004).</li><li>Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+microRNA+dysregulation+in+cancer.">Causes and consequences of microRNA dysregulation in cancer.</a> Nat. Rev. Genet. 10, 704 (2009).</li><li>Coppola, V., De, M. R., Bonci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+prostate+cancer.">MicroRNAs and prostate cancer.</a> Endocr. Relat. Cancer. 17, 1-1 (2010).</li><li>Gordanpour, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-221+Is+down-regulated+in+TMPRSS2:ERG+fusion-positive+prostate+cancer.">miR-221 Is down-regulated in TMPRSS2:ERG fusion-positive prostate cancer.</a> Anticancer Res. 31, 403-403 (2011).</li><li>Nam, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+TMPRSS2:ERG+fusion+gene+predicts+cancer+recurrence+after+surgery+for+localised+prostate+cancer.">Expression of the TMPRSS2:ERG fusion gene predicts cancer recurrence after surgery for localised prostate cancer.</a> Br. J. Cancer. 97, 1690 (2007).</li><li>Ambs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+microRNA+and+messenger+RNA+reveals+deregulated+microRNA+expression+in+prostate+cancer.">Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.</a> Cancer Res. 68, 6162 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-34a+inhibits+prostate+cancer+stem+cells+and+metastasis+by+directly+repressing+CD44.">The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.</a> Nat. Med. 17, 211 (2011).</li></ol>

وقد تم التعبير عن الشاذة من بعض miRNAs وجدت على الدوام في أورام البروستات بالمقارنة مع الأنسجة الطبيعية 10، والذين وردت أسماؤهم بعض هذه miRNAs كعوامل محتملة علاجية جديدة ضد سرطان البروستاتا 11. لذلك نجد أن مستويات التعبير المنحرف من miRNAs تكون مفيدة المؤشرات الحيو...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> TRIzol الكاشف </td><td> إينفيتروجن </td><td> 15596 </td></tr><tr><td> miScript كيت النسخ العكسي </td><td> QIAGEN </td><td> 218061 </td></tr><tr><td> فحوصات التمهيدي miScript </td><td> QIAGEN </td><td> تجربة محددة </td></tr><tr><td> miScript SYBR الأخضر PCR كيت </td><td> QIAGEN </td><td> 218073 </td></tr><tr><td> LightCycler 3.5 نظام PCR في الوقت الحقيقي </td><td> روش </td><td></td></tr><tr><td> ضوء Cycler الأوعية الشعرية </td><td> روش </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 طيفي </td><td> الحرارية العلمية </td><td> 2538 </td></tr><tr><td> اجيلنت 2100 Bioanalyzer </td><td> G2943CA </td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR at JoVE.com

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

الكمية بوليميريز سلسلة من ردود الفعل في الوقت الحقيقي (qPCR) هو وسيلة سريعة وحساسة للتحقيق في مستويات التعبير عن مختلف microRNA (ميرنا) الجزيئات في عينات الورم. ويمكن استخدام هذا التعبير أسلوب مئات من جزيئات ميرنا مختلفة يتم تضخيمها، كميا، وتحليلها من القالب نفسه [كدنا].

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 ...

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Medicine

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

متعدد الأطياف في الوقت الحقيقي التصوير الإسفار عن الكشف عن جراحة داخلية من الحارس العقدة الليمفاوية في امراض النساء والاورام

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A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

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تقييم امتصاص الجسيمات النانوية في الأورام في الوقت الحقيقي باستخدام التصوير Intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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كشف وGenogrouping من Noroviruses من براز الأطفال وذلك في خطوة واحدة Taqman RT-PCR

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تعيين تباين التعبير البروتين في الأورام باستخدام المناعي الكمي

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Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

تحديد<em&gt; الجمال النائم</em&gt; الإدراج ينقول في الأورام الصلبة باستخدام رابط بوساطة PCR

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حركة إعادة التدريب في الوقت الحقيقي باستخدام ملاحظات من الأداء

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Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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النخاعي السوائل عن طريق الرنا الميكروي التنميط الكمي في الوقت الحقيقي PCR

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في الوقت الحقيقي السمسة فحوصات في الدم الكامل بالبشر

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