We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

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The authors have nothing to disclose.

ביטוי גני פרופיל מדגימות אנושיות יכול להיות מאתגר, לא רק לאיכות או כמות הרקמה זמינה, אלא גם לגופים היסטולוגית השונים קיימים בדגימת רקמה נתון. זה מאתגר במיוחד בערמונית שבו רקמות שפירות הן במידה רבה רקמות ואזורים של סרטן סטרומה הן נטולי סטרומה. LCM מאפשר הפרדה פיזית של סטרומה הערמונית ו- RNA האפיתל לחתימה מדויקת יותר של שני סוגי התאים השונים (איור 1 א). בהשוואה לmacrodissection או עיבוד רקמות שלם, LCM יכול להפריד תאי תאים ברקמות שפירות, אך באופן משמעותי יותר יקר ועבודה אינטנסיבית. כמו בכל טכניקה יש מלכודות אפשריות. לדוגמא, איכות RNA יכולה להיות חלקית מושפלת במהלך רכש דגימה ראשוני או במהלך הליך LCM וחילוץ. במקרה הראשון, ביטוי גנים יכולים להיות מושגת על מדידת ampl הקצר שונהסמלים על ידי qPCR. במקרה השני, טיפול חינם קפדן RNase של כל הכלים והחומרים יש לנקוט כדי למנוע השפלה RNA, כמו גם טיפול ועיבוד מדוקדק של סעיפי רקמות. יתר על כן, הגבלת הזמן בילה על שקופית אחת פחות מ 90 דק &#39;שומרת על איכות RNA בתהליך האיסוף. המגבלה הגדולה ביותר של טכניקת LCM היא הגישה למכשיר LCM והיא העבודה הנדרשת כדי לעשות LCM. ישנם מספר סוגים של מיקרוסקופי microdissection לייזר ללכוד זמינים. פרוטוקול זה מתאר שימוש בLCM המשתמש בליזר כדי להותיר את האזורים, שאז נופל לכובע אוסף להלן. סוג זה של LCM הוא אידיאלי להפרדה של האפיתל וסטרומה ברקמת ערמונית אשר מכילה אזורים רב-תאיים בדידים לאסוף, אבל זה סוג של LCM אינו מתאים לתאים בודדים בודדים בידוד מרקמה. לדוגמא, לא אמור לשמש בפרוטוקול זה כדי לאסוף מקרופאג התושב<html> ים, תאים נוירואנדוקריניים או לימפוציטים הסתננות, אשר לעתים קרובות נוכחי כתאים בודדים. יש מכשירים אחרים LCM ושיטות לנצל לייזר ל&quot; לירות &quot;תאים בודדים מהרקמה על כובע קרום, המאפשר בידוד של סוגי תאים אחרים אלה. לא ניתן להתעלם בקרת איכות יסודית ואפיון של RNA. הספיגה ב 260 ננומטר מתאימה לכמת RNA ללימודי RT-qPCR (טבלה 1), אבל לשיטה מבוססת-צבע בצורה מדויקת יותר רצף-רנ&quot;א מכמתת את RNA. נושא חשוב נוסף בניתוח RT-qPCR הוא כדי למנוע הטיה פוטנציאלית מזיהום הדנ&quot;א שייר. זה יכול להיות מושלם על ידי שימוש בצבעי יסוד כי היקף אינטרונים. יש גם ההטרוגניות מטופל גבוהה בביטוי גני סוכנת בית, ולכן שימוש בגני סוכנת בית מרובים הוא הציע ואנחנו משתמשים בדרך כלל 04:57 4,7-9 (איור 1 ב - C וטבלה 2). s = &quot;jove_content&quot;&gt; הבחירה של שיטת פרופיל ביטוי גנים אינו מבוסס רק על הכמות של RNA שנאסף, אלא גם על הסוג של נתונים שהאדם רוצה לרכוש ולהשוות. RT-qPCR הרגיל הוא רגיש יותר מרצף העמוק של הדור הבא, ודורש כמות קטנה יחסית של RNA וכך להקטין את זמן הליך LCM 4. רצף RNA של הדור הבא הוא טכניקה יעילה לכמת את הביטוי הן למיני RNA קצרים וארוכים. רצף של הדור הבא גם מאפשר גילוי של מיני RNA רומן 13. למרבה המזל, את העלות של רצף הדור הבא נמצאת במגמת ירידה. לסיכום, פרוטוקול זה מאפשר בידוד RNA של אוכלוסיות הומוגניות של תאי האפיתל או סטרומה מרקמת ערמונית קפוא. RNA יכול לשמש לטכניקות ניתוח במורד הזרם רבות כדי לספק כלי לביטוי גנים ספציפי לסוג תא מדויק בערמונית שפירה וחולה. סוג זה של נתונים תורם לידיעההבנה של איך ד ביטוי RNA שונה בפתולוגיה מחלה.

בלוטת הערמונית היא רקמה הטרוגנית מורכבת מאפיתל ההפרשה מסודר בacini בלוטות מוקפים stroma fibromuscular מורכב בעיקר של שריר חלק 1. תא אפיתל מורכב מחמישה סוגי תאים שונים, אך מאורגנים: תאי בסיס, תאי הפרשה, תאים נוירואנדוקריניים, תאי הגברת מעבר ותאי גזע 2. בסרטן הערמונית (PCA), הנובע מתאי אפיתל luminal, הצמיחה של אדנוקרצינומה גורמת ירידה הדרגתית מאליי של תא סטרומה 3. מסיבות אלה, יהיו דגימות רקמת הבדלים ברורים בשיעור של סטרומה וסוג תא אפיתל המבוסס על היקף PCA. הבדלים אלה יכולים להוביל להנחות מגמתיות של נתונים ביטוי גנים שנרכשו מכל רקמות ללא קשר לmicrodissection של סוג התא הרצוי. לכן, כדי להסיר הטיה זו היא חיוני על מנת להפריד בין סוגי תאים לפני מיצוי RNA וניתוח ביטוי גנים. Macrodissection או microdissection ניתן להשתמש כדי להפריד פיזי אזורי אפיתל מאופיינים היטב מסטרומה סביב 4 -6. Macrodissection נעשה בדרך כלל בסכין גילוח תחת מיקרוסקופ לנתח ועובד היטב להפרדת PCA הגדולה גושים מסטרומה, אבל הוא לא מסוגל להסיר אפיתל השפיר מסביב סטרומה (ראה דוגמא של היסטולוגיה ערמונית שפירה באיור 1). Microdissection עם לייזר (LCM) הוא באופן משמעותי יותר מאשר עבודה אינטנסיבי macrodissection, אבל מאוד מדויק יכול לנתח אפיתל השפיר 4. הפרסומים אחרונים מהמעבדה שלנו הראו כי RNA ניתן לחלץ בהצלחה על ידי LCM משתי ביופסיות-קבוע פורמלין מוטבע פרפין (FFPE) או רקמה קפוא 4,7-9. האתגרים המרכזיים בחילוץ LCM-רנ&quot;א הם 1) לנתח במדויק את האזורים הרצויים של הרקמה, ו- 2) לשמר Rשלמות NA במהלך 4,10 תהליך LCM והבידוד. RNA מבודד מאוכלוסיות תאים טהורות יכול לשמש לניתוח ביטוי גנים על ידי מספר שיטות כולל הפוך-שעתוק PCR כמותי (RT-qPCR) 7,8, microarray 11, ו-sequencing העמוק 12-14. המטרה של פרוטוקול זה היא לבודד RNA הכולל מהאפיתל של הערמונית LCM מהרקמה קפוא לביטוי גנים במורד הזרם מנתח.

<table><tbody><tr><td>RNase-AWAY&amp;nbsp;</td><td>MBP</td><td>7005-11</td><td></td></tr><tr><td>PEN-membrane 4.0 mm slides&amp;nbsp;</td><td>Leica</td><td>11600289</td><td></td></tr><tr><td>Glass slides, Superfrost Plus</td><td>FisherBrand</td><td>12-550-15</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon labs.</td><td>2701</td><td></td></tr><tr><td>DEPC (diethyl pyrocarbonate)</td><td>Sigma</td><td>D-5758</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050</td><td></td></tr><tr><td>Coplins (Staining jar)</td><td>IHCWORLD</td><td>M900-12</td><td></td></tr><tr><td>Coplins (Staining rack)</td><td>IHCWORLD</td><td>M905-12</td><td></td></tr><tr><td>Aperio ScanScope</td><td>Aperio(Leica)</td><td>ScanScope&amp;reg; CS</td><td></td></tr><tr><td>Toluidine Blue</td><td>Fluka</td><td>89640-5G</td><td></td></tr><tr><td>Laser Microdissection System</td><td>Leica</td><td>LMD7000</td><td></td></tr><tr><td>0.5 ml Thin-walled Tubes for LCM</td><td>Thermo Scientific</td><td>AB-0350</td><td></td></tr><tr><td>RNAqueous&amp;reg;-Micro Total RNA Isolation Kit</td><td>Ambion</td><td>AM1931</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Life Technologies</td><td>Q32866</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368814</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Universal cDNA Synthesis Kit II, 8-64 rxns</td><td>Exiqon</td><td>203301</td><td></td></tr><tr><td>TaqMan microRNA RT kit</td><td>Applied Biosystems</td><td>4366597</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Hematoxylin stain&amp;nbsp;</td><td>Ricca Chemical Company</td><td>3536-16</td><td></td></tr><tr><td>Eosin-Y</td><td>Richard Allan Scientific&amp;nbsp;</td><td>7111</td><td></td></tr></tbody></table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

במחקר קודם שהראינו את השימוש של LCM לאסוף רקמות אפיתל וסטרומה להשוות פרופיל ביטוי על ידי RT-qPCR של mRNA ומייקרו RNA מרקמות ערמונית קפוא וFFPE מאותו החולה 4. LCM זמן רב, במיוחד אם כמות גדולה של RNA היא שתיגבה עבור ניתוח רצף של הדור הבא. לכן, חשוב לשמור על מרחב העבודה והכלים RNase חופשיים. מומלץ לבחון בקרות איכות ותא-סגוליות בRNA שנאסף (דן בפירוט רב יותר בסעיף הבא). איור 1 מציג קטע ערמונית מוכתם בשקופית PEN-מסגרת מתחת למיקרוסקופ לפני ואחרי אפיתל השפיר לכידה-הלייזר . לאחר המדגם נאסף וRNA מופק בקפידה, חשוב להעריך את האיכות וכמות RNA כפי שמוצג בטבלה 1. אין זה נדיר להתבונן 260/280 יחסי ננומטר נמוכים. ריכוז RNA יהיהלשפר את יחסי 260/280 ננומטר, אבל זה יכול גם לגרום לאובדן של מיני RNA קטנים. בנוסף לבקרת איכות, חשוב לבחון בקרות סוג ספציפי תא כדי לאשר את הספציפיות של האזור-נתפס הלייזר של הרקמה (איור 1 ב - C). לדוגמא, באיור 1 הביטוי של גן AMACR נמדד כדי לאשר רקמת האפיתל בסרטן הערמונית בהשוואה לרקמת האפיתל נורמלית ,. באיור 1C הביטוי של NKX3.1 אישר העדר סטרומה במדגם-נאסף LCM. האיכות של RNA בדגימה גם יכולה להיות compareed לRNA של ידוע, RNA באיכות טובה. ערכי RT-qPCR Ct לRNAs מבודד מרקמת LCM-collected- נמצאים באותו הטווח כמו RNA שנאסף מתאי האפיתל עיקריים תרבותיים, המאשר את איכות של RNA הארוך וקטן שחולץ (טבלה 2). לבסוף, הטבלה 3 מראה כי RNA זה הוא באיכות מספיק לsequenci RNA של הדור הבאng. <img alt="איור 1" src="/files/ftp_upload/53405/53405fig1.jpg" /> איור 1: אוסף של האפיתל שפיר, האפיתל סרטן הערמונית וסטרומה מערמונית אנושית על ידי מיקרו לנתיחה לייזר ללכוד ביופסיה של ערמונית () מוכתמת בtoluidine הכחול לפני ואחרי LCM-אוסף של האפיתל השפיר.. (ב - ג) רקמות שנאספו על ידי LCM מ -45 חולים להביע סמנים גנטיים מתאימים של זהות סלולרית 7. AMACR (B) הוא סמן של סרטן הערמונית וניתן לזהות רק ברקמות סרטן. NKX3.1 (ג) הוא סמן של האפיתל ולא להבחין ברקמות סטרומה. נתון זה הוא שונה מGiangreco et al., JSBMB 2,015 7. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53405/53405fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="שולחן 1" src="/files/ftp_upload/53405/53405tab1.jpg" /> טבלת 1: איכות וכמות RNA מרקמת ערמונית שנאסף LCM. <img alt="טבלה 2" src="/files/ftp_upload/53405/53405tab2.jpg" /> טבלה 2: RT-qPCR מאפיתל השפיר LCM נאסף הערמונית. <img alt="טבלה 3" src="/files/ftp_upload/53405/53405tab3.jpg" /> טבלה 3: RNAseq מרקמת ערמונית שנאסף LCM.

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Microdissection לייזר לכידתו של אדם הערמונית האפיתל לניתוח RNA

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.4K Views.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Video: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

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 ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

רצפי RNA קטן ללא קידוד מתוך פורמאלין-רקמות קבועות וסרום-אקסוזומים נגזרות מחולי סרטן הערמונית עמידים סירוס

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Cancer Research

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

זיהוי מבוסס microarray של פרט HERV לוקוסים ביטוי: בקשה לגילוי סמן ביולוגי בסרטן הערמונית

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

לכידת לייזר מיקרו חיתוך של העכבר סחוס ועצם עבור ניתוח ביטוי גנים

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Developmental Biology

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, ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

אופטימיזציה של לכידת לייזר Microdissection עבור בידודו של הגרעינים המעית מרקמות אנושיות מוקפאות

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

בידוד RNA מן אוכלוסיות תאים ספציפיות באמצעות Microdissection לייזר ללכוד בשילוב עם ראפיד immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Biology

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

לייזר Microdissection לכידה של מאוד טהור Meshwork Trabecular מעיני העכבר עבור ניתוח ביטוי גנטי

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

לכידת לייזר פרוטוקול מיקרודיסקציה התשואות באיכות גבוהה RNA מ-העכבר קפואים טרי-קפוא

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

יישום של מיקרו-דיסקציה בלייזר לחשיפת תעתיקים אזוריים ברקמת הכליה האנושית

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...