אנו מודים ט האנג, MC. צאי, א &#39;מנור, א&#39; סגל, מ &#39;קארודה, ט Swigut, ואני Shestopalov לדיונים. נתמך על ידי הסוכנות למדע, טכנולוגיה ומחקר של סינגפור (CC), NIH R01-R01 ו-CA118750 HG004361 (HYC), וקליפורניה המכון לרפואה הרגנרציה (HYC). HYC הוא המדען הקריירה המוקדמת של הווארד יוז רפואי במכון.

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rox1+and+rox2+RNAs+are+essential+components+of+the+compensasome,+which+mediates+dosage+compensation+in+Drosophila.">The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila.</a> Mol. Cell. 4, 117-122 (1999).</li><li>Gupta, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+HOTAIR+reprograms+chromatin+state+to+promote+cancer+metastasis.">Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.</a> Nature. 464, 1071-1076 (2010).</li><li>Tsai, M. 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ס צ &#39;ו ח.י. צ&#39;אנג נקראים כמו ממציאים על יישום פטנט על שיטה זו.

כאן תיארנו ציוץ-seq, שיטת מיפוי באתרים ב vivo lncRNA מחייב הגנום כולו. הפרמטרים העיקריים להצלחה הם בריכות מפוצלים של ריצוף בדיקות oligonucleotide ו crosslinking glutaraldehyde. העיצוב של זיקה הבדיקות, הוא פשוט נתן את רצף הרנ&quot;א ואינה דורשת ידע מוקדם על מבנה של RNA או תחומים פונקציונליים. ההצלחה שלנו עם roX2, TERC, ו HOTAIR - שלושה RNAs שונות למדי בשני המינים - מצביע על כך ציוץ-seq היא להכליל צפוי lncRNAs רבים. כמו עם כל הניסויים, פקדי טיפול ונכון נדרשים לפרש את התוצאות. LncRNA שונים עשויים לדרוש טיטרציה של התנאים, ושינוי מושכל של תנאים, כגון מבחר של בדיקות קרבה או crosslinkers שונים, עשוי להדגיש היבטים שונים של RNA-הכרומטין אינטראקציות. כמו SEQ-Chip, לא כל האירועים המחייבים הינם פונקציונלי בהכרח, ועל מחקרים נוספים נדרשים כדי לברר את ההשלכות הביולוגיות של RNA Occupancy על הכרומטין. עם זאת, אנו צופים יישומים מעניינים של טכנולוגיה זו עבור חוקרים של הכרומטין הקשורים lncRNAs אחרים, אשר כעת מספר אלפי 8,9. בדיוק כמו שבב ואילך פתחה את הדלת הגנום כולו החקירות של אינטראקציות חלבון דנ&quot;א, ציוץ-seq מחקרים של &quot;RNA interactome&quot; עשוי לחשוף אפיקים חדשים רבים של הביולוגיה.

<table><tbody><tr><td colspan="4">Buffer List:</td></tr><tr><td colspan="4">Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20 &amp;deg;C.</td></tr><tr><td colspan="4">Lysis Buffer:</td></tr><tr><td colspan="4">50 mM Tris-Cl pH 7.0&lt;br/&gt;10 mM EDTA&lt;br/&gt;1% SDS&lt;br/&gt;Always add PMSF, P.I. and Superase-in fresh before use except when washing beads</td></tr><tr><td colspan="4">Proteinase K Buffer (for DNA)</td></tr><tr><td colspan="4">100 mM NaCl&lt;br/&gt;10 mM TrisCl pH 8.0 (For RNA use pH 7.0)&lt;br/&gt;1 mM EDTA&lt;br/&gt;0.5% SDS&lt;br/&gt;Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use</td></tr><tr><td colspan="4">Hybridization Buffer</td></tr><tr><td colspan="4">750 mM NaCl&lt;br/&gt;1% SDS&lt;br/&gt;50 mM Tris-Cl pH 7.0&lt;br/&gt;1 mM EDTA&lt;br/&gt;15% formamide (store in the dark at 4 &amp;deg;C)&lt;br/&gt;Always add PMSF, P.I. and Superase-in fresh before use</td></tr><tr><td colspan="4">Wash Buffer</td></tr><tr><td colspan="4">2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)&lt;br/&gt;0.5% SDS&lt;br/&gt;Always add PMSF fresh before use</td></tr><tr><td colspan="4">DNA elution Buffer</td></tr><tr><td colspan="4">50 mM NaHCO3&lt;br/&gt;1% SDS</td></tr><tr><td colspan="4">Table of specific reagents and equipment:</td></tr><tr><td>Glutaraldehyde (EM grade)</td><td>Sigma-Aldrich</td><td>G5882-10x10ml</td><td></td></tr><tr><td>Motorized pellet mixer</td><td>VWR international</td><td>V8185-904</td><td></td></tr><tr><td>Protease inhibitor</td><td>Roche Group</td><td>11873580001</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>78830</td><td></td></tr><tr><td>Superase-in</td><td>Ambion</td><td>&lt;a href=&quot;http://www.ambion.com/catalog/CatNum.php?AM2696&quot;&gt;AM2696&lt;/a&gt;</td><td></td></tr><tr><td>Bioruptor</td><td>Diagenode</td><td>UCD-200</td><td></td></tr><tr><td>Falcon tubes (for sonication)</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>Proteinase K</td><td>Ambion</td><td>AM2546</td><td></td></tr><tr><td>PCR purification kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>C-1 magnetic beads</td><td>Invitrogen</td><td>65002</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626-25G</td><td></td></tr><tr><td>DynaMag-15 magnet</td><td>Invitrogen</td><td>123-01D</td><td></td></tr><tr><td>DynaMag-2 magnet</td><td>Invitrogen</td><td>123-21D</td><td></td></tr><tr><td>MIRNeasy mini kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Rnase H</td><td>Epicentre Biotechnologies</td><td>R0601K</td><td></td></tr><tr><td>Rnase A</td><td>Sigma-Aldrich</td><td>R4875-100MG</td><td></td></tr><tr><td>Phase Lock Gel Heavy</td><td>5 PRIME</td><td>2302810</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>Phenol:chloroform:Isoamyl</td><td>Invitrogen</td><td>15593-031</td><td></td></tr><tr><td>Chloroform</td><td>Ricca</td><td>RSOC0020-1C</td><td></td></tr><tr><td>GlycoBlue</td><td>Ambion</td><td>AM9515</td><td></td></tr><tr><td>Glycine</td><td>JT Baker</td><td>4057-06</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Invitrogen</td><td>10010-049</td><td></td></tr><tr><td>Elution Buffer (EB)</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>20x SSC</td><td>Invitrogen</td><td>15557-036</td><td></td></tr><tr><td>10% SDS</td><td>Invitrogen</td><td>15553-027</td><td></td></tr><tr><td>DNA-&lt;em&gt;free&lt;/em&gt;</td><td>Ambion</td><td>AM1906</td><td></td></tr><tr><td>Buffer kit</td><td>Ambion</td><td>AM9010</td><td></td></tr><tr><td>Formamide</td><td>Invitrogen</td><td>15515-026</td><td></td></tr></tbody></table>

chromatin isolation by rna purification (chirp)

RNAs noncoding ארוכים הם וסתים מרכזיים של הכרומטין מדינות תהליכים ביולוגיים חשובים כגון פיצוי המינון, החתמה, ועל ביטוי גנים התפתחותיים 1,2,3,4,5,6,7. התגלית האחרונה של אלפי lncRNAs בשיתוף עם ספציפיים שינוי מתחמי הכרומטין, כגון קומפלקס מדכא Polycomb 2 (PRC2) כי מתווך היסטון H3 ליזין 27 trimethylation (H3K27me3), מציע תפקידים רחבים עבור lncRNAs רבים בניהול מדינות הכרומטין של הגן הספציפי אופנה 8,9. בעוד כמה lncRNAs נחשבים לעבוד בחבר העמים על גנים סמוכים, lncRNAs אחרים לעבוד טרנס להסדיר גנים הממוקמים רחוק. למשל, תסיסנית lncRNAs roX1 ו roX2 לאגד באזורים רבים על כרומוזום X של תאים זכריים, הם קריטיים עבור פיצוי במינון 10,11. עם זאת, המיקומים המדויקים של אתרי הקישור שלהם אינם ידועים ברזולוציה גבוהה. באופן דומה, אדם lncRNA HOTAIR יכול להשפיע PRC2 תפוסה על Hundreds של גנים בגנום כולו 3,12,13, אבל איך הספציפיות מושגת אינו ברור. LncRNAs יכול גם לשמש פיגומים מודולריים לגייס את הרכבה של קומפלקסים חלבונים רבים. קלאסי חוצה משחק RNA הפיגום הוא RNA TERC המשמש תבנית ואת הפיגום על telomerase מורכב 14; HOTAIR יכול גם לשמש פיגום עבור PRC2 ו demethylase H3K4 מורכב 13. מחקרים קודמים מיפוי תפוסה RNA על הכרומטין חשפו תובנות משמעותיות 15,16, אבל רק לוקוס גן יחיד בכל פעם. האתרים התפוסה של רוב lncRNAs אינם ידועים, וכן את התפקידים של lncRNAs בוויסות הכרומטין היה להסיק בעיקר את ההשפעות העקיפות של ההפרעות lncRNA. כשם immunoprecipitation הכרומטין ואחריו microarray או רצף עמוק (שבב שבב או שבב seq, בהתאמה) השתפר מאוד את הבנתנו-DNA חלבון אינטראקציות בקנה מידה גנומי, הנה אנחנו להמחיש recenפורסם tly האסטרטגיה למפות רב תפוסה RNA הגנום כולו ברזולוציה גבוהה 17. , שיטה זו בידוד הכרומטין על ידי טיהור RNA (ציוץ) (איור 1), מבוסס על לכידת הזיקה של יעד lncRNA: מורכב הכרומטין על ידי ריצוף antisense-oligos, אשר לאחר מכן יוצרת מפה של אתרי הקישור הגנומי ברזולוציה של מאות בסיסים שונים עם רגישות גבוה ונמוך רקע. ציוץ הוא ישים lncRNAs רבים בגלל העיצוב של זיקה הבדיקות, הוא פשוט נתן את רצף הרנ&quot;א ואינה דורשת ידע על מבנה של RNA או תחומים פונקציונליים.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

ציוץ הוא טכניקה חדשנית ומהירה למפות אתרי הקישור הגנומי של RNAs noncoding ארוכות (lncRNAs). השיטה מנצלת את הייחודיות של תחושת אנטי oligonucleotides ריצוף כדי לאפשר ספירה של lncRNA הנכנס אתרים הגנומי.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

86.1K Views.  Stanford University School of Medicine. ציוץ הוא טכניקה חדשנית ומהירה למפות אתרי הקישור הגנומי של RNAs noncoding ארוכות (lncRNAs). השיטה מנצלת את הייחודיות של תחושת אנטי oligonucleotides ריצוף כדי לאפשר ספירה של lncRNA הנכנס אתרים הגנומי.

Video: Chromatin Isolation by RNA Purification ChIRP

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

בידוד של קומפלקסים RNA חלבון מאותו מקור מתאי שימוש Elution oligonucleotide מכוונת

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Biochemistry

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

תא בודד לשימוש חוזר לניתוחים אפיגנומיים איטרטיביים

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

Genetics

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

<meta charset="utf8"></head><body>בידוד ועיבוד של אדיפוציטים לבנים של עכברים לניתוח טרנסקריפטום ואפיגנום

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

בידוד ועיבוד של אדיפוציטים לבנים של עכברים לניתוח טרנסקריפטום ואפיגנום

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

הכנת RNA ארוך ציטופלזמי וגרעיני מתאים ראשוניים ותאי תרבית

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how ...

Cancer Research

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

הכרומטין ניתוח אינטראקציה עם מנבא סוף תג הרצף (Chia-PET) עבור מיפוי אינטראקציות הכרומטין הבנת תקנה תמלול

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

שיטה לבידוד וזיהוי mRNAs, רנ&quot;א ורכיבי חלבון של מכלולי Ribonucleoprotein מתמציות סלולריות באמצעות שבב RIP

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

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 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

בידוד RNA מן העכבר לבלב: רקמות ribonuclease עשיר

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

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בידוד של קומפלקסים RNA חלבון מאותו מקור מתאי שימוש Elution oligonucleotide מכוונת

תא בודד לשימוש חוזר לניתוחים אפיגנומיים איטרטיביים

בידוד ועיבוד של אדיפוציטים לבנים של עכברים לניתוח טרנסקריפטום ואפיגנום

בידוד ועיבוד של אדיפוציטים לבנים של עכברים לניתוח טרנסקריפטום ואפיגנום

הכנת RNA ארוך ציטופלזמי וגרעיני מתאים ראשוניים ותאי תרבית

הכרומטין ניתוח אינטראקציה עם מנבא סוף תג הרצף (Chia-PET) עבור מיפוי אינטראקציות הכרומטין הבנת תקנה תמלול

שיטה לבידוד וזיהוי mRNAs, רנ"א ורכיבי חלבון של מכלולי Ribonucleoprotein מתמציות סלולריות באמצעות שבב RIP

בידוד של קטן noncoding RNAs מסרום האדם

בידוד RNA מן העכבר לבלב: רקמות ribonuclease עשיר

בידוד של אזורים הגנומי ספציפיים וזיהוי של Associated מולקולות על ידי enChIP