אנו מודים אן דה יונג וSiger Holsappel לעזרה עם הפקת שקופיות מערכי DNA. תמיכתה של אנה דה יונג לניתוח ביואינפורמטיקה גם מוערכת. אנו מודים גם Jelle Slager לבחינת הנייר. מוחמד אפזאל ואירפאן Manzoor נתמך על ידי אוניברסיטת GC, Faisalabad, פקיסטן במסגרת תכנית פיתוח סגל של HEC פקיסטן.

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המחברים מצהירים שאין להם אינטרסים כלכליים מתחרים.

אנו מתארים פרוטוקול ידידותי למשתמש שניתן ליישם כדי לבצע ניתוח transcriptome שלם של חיידקים. נקודת המפתח על הטכניקה המסוימת הזה היא שהמצב לפיו התאים נקצרים ישתנה. לאחר קצירת תאי הבידוד ו- RNA, טכניקה זו הופכת להיות שווה לכל סוגי דגימות חיידקים ופועלת בהתאם לשלבים זהים לחלוטין...

המחקר של המערך השלם של mRNA שפע (transcriptome) מקודד על ידי הגנום של האורגניזם חד-תאי או תא האיקריוטים בזמן מסוים או במצב מסוים, ובכלל זה ביטוי יתר של גן או נוק-אאוט, נקרא transcriptomics. Transcriptomics מאפשר לנו להתבונן למה גני המידה באים לידי ביטוי במצב מסוים בנקודת זמן X ונותן לנו מידע על כמה חזק גנים באים לידי ביטוי ביחס להתייחסות. Microarray הוא מערך דו-ממדי על מצע מוצק (בדרך כלל שקופיות זכוכית או תא סרט דק סיליקון) שיכול לשמש לassay כמויות גדולות של חומר ביולוגי באמצעות הקרנת תפוקה גבוהה, וממוזער, עיבוד מרובב ומקביל וזיהוי שיטות. Microarrays בא בסוגים שונים, כולל DNA-microarrays, microarrays חלבון, microarrays פפטיד, microarrays רקמות, microarrays נוגדן, microarrays הסלולרי ואחרים. מערך DNA הואבעצם הרכבה של כתמים מיקרוסקופיים DNA קבוע למשטח מוצק, בדרך כלל זכוכית. מערכי DNA משמשים למדידת רמות הביטוי של גן או קבוצת גנים בו זמנית או לגנוטיפ אזורים מרובים של הגנום 2,3. Picomoles (10 -12 שומות) של חללית נמצא בתוך כל מקום DNA שמייצג רצף DNA ספציפי, הידוע גם בכתב. מולקולות mRNA שכותרתו מהדגימות נקראות &#39;יעדים&#39;. Fluorophores משמשות למדידת הכלאת בדיקה-יעד וזיהוי של מטרות שכותרתו fluorophore קובע את השפע היחסי של רצפי חומצות גרעין ביעד. ניסוי microarray יכול להשיג בדיקות גנטיות מרובות במקביל כי מערך עשוי להכיל עשרות אלפי בדיקות. הפריסה של ניסוי microarray פשוט מוצגת באיור 1. לאחרונה, הוקם במעבדות האחרות שלנו ושהמערכים אלה הם לשימוש חוזר, מה שהופך את עלות effectiv די בטכניקה זודואר. טכניקות בידוד RNA וטיהור שונות פותחו לאורך השנים כולל C-TAB, SDS ושיטות GT 4-8. יתר על כן, כמה ערכות מסחריות זמינות גם. לביטוי גני RNA באיכות גבוהה הוא חשוב מאוד. לכן, שיטות בידוד RNA הן שונה כדי לקבל כמות המרבית של RNA. באופן דומה, הצעדים להכנת cDNA וסימון של cDNA הם מזעריים. נורמליזציה של נתונים לאחר הסריקה מבוצעת גם ביעילות על ידי שימוש בחבילות תוכנה ב- בית שנבנה וכלים 9. pneumoniae Streptococcus הוא הפתוגן אנושי גרם-חיובי שcolonizes לוע האף והוא הגורם לזיהומים רבים כגון דלקת ריאות, אלח דם, דלקת אוזן תיכונה ודלקת קרום המוח 10. החיידק יכול לנצל את מגוון רחב של החומרים המזינים הדרושים לצמיחה והישרדות 11,12. מספר המחקרים בוצעו עלחילוף חומרים דלקת ריאות חנקן ורגולציה המדגישים את החשיבות של חומצות אמינו ותפקידם בארסיות 13,14. במחקר זה, תגובת transcriptomic של ס &#39; pneumoniae לשינוי ריכוזים של L-סרין, חומצת אמינו בשפע קיים בפלזמת הדם האנושית, הוא דיווח באמצעות מערכי DNA. תגובת transcriptomic של ס &#39; pneumoniae גדל בריכוז מינימאלי של L-סרין (150 מיקרומטר) הושווה שלגדל בריכוז מרבי (10 מ&quot;מ) של סרין. בינוני הגדרה כימית (CDM או בינוני מינימאלי) 15 שימש למחקר זה כדי לשלוט בריכוז של סרין. המוקד של מחקר זה הוא להפוך ידידותי למשתמש בטכניקה זו ולספק כלים שונים לנורמליזציה נתונים וניתוח. לכן, מספר הכלים שפותחו לצורך הניתוח ופרשנות הנתונים. FIVA (מידע Viewer פונקציונלי וAnalyzer) מספק פלטפורמה לעיבוד המידע הכלול באשכולות של גנים שישדפוסים דומים ביטוי גנים ולבניית פרופילים תפקודיים 16. התובע הוא חבילת תוכנה אחרת המאפשרת זיהוי של פונקציות והסברים של גנים 17 משוערים. על ידי עשיית שימוש של שיטות קיבוץ באשכולות, לחשוף מספק אלגוריתם זיהוי אתר הקישור DNA. המוטיבים רגולטורית הסיס של גנים יכולים להיות מוקרנים באמצעות אלגוריתם זה 18. Genome2D מציע פלטפורמה מבוססת Windows להדמיה וניתוח של נתונים transcriptome על ידי הצעת טווחי צבע שונים כדי לאפיין את השינויים ברמות ביטוי גנים על מפת הגנום 19. שרת האינטרנט של הפלפל מציע, בנוסף לשיטת ניתוח all-in-one, ארגז כלים לכרייה לregulons, יזמים וגורם שעתוק מחייב אתרים 20. ביאור מלא של אזורים חוץ-בגנום של חיידקים יכול להיות מושגת על ידי שימוש בחבילה זו. ביולוגים יכולים להפיק תועלת רבה מפלפל כפי שהוא מציע להם במה לעיצוב ניסויs כך שהמידע המשוער ניתן לאשר במבחנה 20. חבילות תוכנה אלה תורמים באופן משמעותי לניתוח microarray כמו רובם זמינים באופן חופשי ולעשות נורמליזציה נתונים וניתוח אמינה מאוד.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acid phenol</td>
			<td>SigmaAldrich</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr>
			<td>Roche RNA isolation kit</td>
			<td>Roche Applied Science</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td></td>
			<td>105015</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Boom</td>
			<td>92013505.1000.</td>
			<td></td>
		</tr>
		<tr>
			<td>IAA</td>
			<td></td>
			<td>106630</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Nanodrop</td>
			<td>ND-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent BioAnalyser</td>
			<td>Agilent</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>AA-dUTP</td>
			<td>Life technologies Invitrogen</td>
			<td>AM8439</td>
			<td></td>
		</tr>
		<tr>
			<td>DDT</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>First Strand buffer</td>
			<td>Life technologies Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SigmaAldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight-550</td>
			<td>Thermoscientiffic</td>
			<td>62262</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight-650</td>
			<td>Thermoscientiffic</td>
			<td>62265</td>
			<td></td>
		</tr>
		<tr>
			<td>SHY Buffer</td>
			<td>SigmaAldrich</td>
			<td>H7033-125ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedvac cooler</td>
			<td>Eppendorf</td>
			<td>RUGNE3140</td>
			<td>Speedvac concentrator plus</td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Grant Boekel</td>
			<td>Iso-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Lifter-slips</td>
			<td>Erie Scientific</td>
			<td>25x60I-M-5439</td>
			<td></td>
		</tr>
		<tr>
			<td>Wipe</td>
			<td>KIMTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>SigmaAldrich</td>
			<td>L3771-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC</td>
			<td>SigmaAldrich</td>
			<td>W302600-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Genpix autoloader 4200A1</td>
			<td>MSD analytical technologies</td>
			<td></td>
			<td>Microarray scanner</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>SigmaAldrich</td>
			<td>104766</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fast and reliable pipeline for bacterial transcriptome analysis case study: serine-dependent gene regulation in streptococcus pneumoniae

ביטוי גנים והרגולציה שלה הם מאוד חשובים להבין את התנהגותם של תאים בתנאים שונים. טכניקות שונות משמשות כיום ללמוד ביטוי גנים, אך רובם מוגבלים במונחים של מתן תמונה כוללת של הביטוי לכל transcriptome. מערכי DNA מציעים טכנולוגית מחקר מהירה וכלכלית, אשר נותנת סקירה מלאה של ביטוי גנים הגלובלי ויש להם מספר עצום של יישומים, כולל זיהוי של גנים חדשים וגורם שעתוק מחייבים אתרים, אפיון של פעילות תעתיק של התאים וגם לעזור בניתוח אלפים של גנים (בניסוי יחיד). במחקר הנוכחי, את התנאים לניתוח transcriptome חיידקים מקציר תא לניתוח מערך DNA כבר מותאמים. אם ניקח בחשבון את הזמן, עלויות ודיוק של הניסויים, פלטפורמה טכנולוגית זו מוכיחה להיות שימושי מאוד ואוניברסלי applicabale ללימוד transcripto חיידקיםmes. כאן, אנו מבצעים ניתוח מערך DNA עם pneumoniae Streptococcus כמקרה מבחן-ידי השוואת תגובות תעתיק של ס &#39; pneumoniae גדל בנוכחות ריכוזים שונים L-סרין במדיום. RNA סה&quot;כ היה מבודד באמצעות שיטת Macaloid באמצעות ערכת בידוד RNA והאיכות של RNA נבדק על ידי שימוש בערכת בדיקת איכות RNA. cDNA הוכן באמצעות transcriptase ההפוך ודגימות cDNA תויגו באמצעות אחת משני צבעי ניאון אמין-reactive. שקופיות מערך DNA תוצרת בית שמשו לכלאה של דגימות cDNA המסומנים ונתונים microarray נותחו באמצעות מסגרת עיבוד מראש נתונים microarray cDNA (Microprep). לבסוף, Cyber-T שימש כדי לנתח את הנתונים שנוצרו באמצעות Microprep לזיהוי גנים הביעו באופן דיפרנציאלי משמעותיים מבחינה סטטיסטית. חבילות תוכנה כמו כן, נבנו בבית (פלפל, FIVA, לחשוף, התובע, Genome2D) שמשו כדי לנתחנתונים.

RNA, בידודי cDNA וניתוח L-סרין הוא אחד מחומצות אמינו החיוניות וריכוזו בפלזמת דם אדם משתנה 60-150 מיקרומטר בילדים ומבוגרים. תפקידה בביוסינתזה של purines וpyrimidines מדגיש את חשיבותה בחילוף חומרים והוא מבשר על כמה חומצות אמינו (גליצין, צ...

Watch this Scientific Journal Video about A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae at JoVE.com

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages.

צינור מהיר ואמין למחקר בקטריאלי transcriptome ניתוח מקרה: ויסות גני Serine תלויה ב<em&gt; Streptococcus pneumoniae</em

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays ...

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A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

10.3K Views.  University of Groningen. This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages. 

Video: A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

Bacterial Gene Expression Analysis Using Microarrays

Gene Expression חיידקית ניתוח שימוש microarrays

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

הבידוד של הריבוזום פוליפפטידים המתהווה כרוכים<em&gt; במבחנה</em&gt; כדי לזהות אתרים השהה של תרגום לאורך ה-mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

באמצעות רצף מעצר SecM ככלי לבודד פוליפפטידים כרוכים הריבוזום

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

מחיקות Gene הגנום ב<em&gt; סטרפטוקוקוס sanguinis</em&gt; על ידי תפוקה הגבוהה PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

ניתוח של גלובל RNA סינתזה ברמת התא בודד הבאות היפוקסיה

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

הפקת חומצות גרעין יעיל 16S rRNA ג&#39;ין רצף עבור בקטריאלי קהילת אפיון

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

גישות גנטיות ביוכימיים עבור<em&gt; In vivo</em&gt; ו<em&gt; במבחנה</em&gt; הערכת oligomerization חלבון: מקרה המבחן קולטן Ryanodine

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus on one or more gene families that are significant to the organism and study. In this protocol, we outline bioinformatic steps to generate a phylogeny and to quantify the expression of genes of interest. Phylogenetic trees can give insight into how genes are evolving within and between species as well as reveal orthology. These results can be enhanced using RNA-seq data to compare the expression of these genes in different individuals or tissues. Studies of molecular evolution and expression can reveal modes of evolution and conservation of gene function between species. The characterization of a gene family can serve as a springboard for future studies and can highlight an important gene family in a new genome or transcriptome paper.

The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data.

צינור ביואינפורמטיקה לחקירת אבולוציה מולקולרית וביטוי גנים באמצעות RNA-seq

Distilling and reporting large datasets, such as whole genome or transcriptome data, is often a daunting task. One way to break down results is to focus ...