C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

הערה: רנ&quot;א הכל מ 6 רמה כוללת rRNA, mRNA, ו RNA קטנים אחרים. מאז RNA ריבוזומלי יש מ שופע 6 שינויים A, מדידת מ 6 הבגרויות תצטרך לשקול את העובדה הזו. בידוד RNA 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות פתרון טיהור ribonuclease (מרוסס על מגבות נייר), לנגב את טפטפות ומשטחי ספסל מעבדה. מגבות נייר רטובות עם מי nuclease ללא ולנגב טפטפות ומשטחי ספסל במעבדה שוב. </li><li style=";text-align:right;direction:rtl"> הפקת RNA <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בידוד RNA סה&quot;כ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שימוש בוחש עילי, homogenize 50-100 מ&quot;ג של רקמה או 5-10 x 10 6 תאים 1 מ&quot;ל של תמיסת בידוד RNA שמרו על 4 מעלות צלזיוס. הערה: רקמה יותר (100-150 מ&quot;ג) עשויה להידרש דגימות רקמה שומניות מעכברים בגלל ריכוזי RNA הנמוכים בו. דוגמאות שמקורו מן הלב העכבר, כבד, שרירי השלד, ריאות, מוח, מקרופאגים הועסק 4 בעבר. </li><li style=";text-align:right;direction:rtl"> בcubate homogenates מדגם במשך 5 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הוספת 100 μL של 1-bromo-3-chloropropane (BCP) לכל 1 מ&quot;ל של homogenate המדגם. לנער את צינורות בתוקף למשך 15 שניות ולהמשיך לבודד רנ&quot;א הכל על פי הוראות היצרן 24. </li></ol></li><li style=";text-align:right;direction:rtl"> Polyadenylated mRNA טיהור מ- RNA סה&quot;כ מבודד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לטהר mRNA polyadenylated באמצעות ערכות או פרוטוקולים זמינים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנער היטב מחדש להשעות כל אחד מהפתרונות הבאים: פתרון מחייב 2x, אוליגו חרוזי פוליסטירן (DT), והפתרון לשטוף. ודא אוליגו (DT) חרוזים חם לטמפרטורת החדר לפני השימוש. </li><li style=";text-align:right;direction:rtl"> העבר 120 μL של פתרון elution לכל הכנה לתוך צינור וחום עד 70 ° C בתוך גוש חימום. </li><li style=";text-align:right;direction:rtl"> פיפטה עד 500 מיקרוגרם של רנ&quot;א הכל לתוך צינור microcentrifuge. עם nuclease ללא מים, להתאים את עוצמת הקול כדי 250 μL. </li><li style=";text-align:right;direction:rtl"> הוסף 250 μL של פתרון מחייב 2x אל רנ&quot;א הכל כךlution מערבולת בקצרה לערבב את תוכנו. </li><li style=";text-align:right;direction:rtl"> הוסיפו 15 μL של אוליגו (DT) חרוז מערבולת ביסודיות לערבב. </li><li style=";text-align:right;direction:rtl"> דגירה את התערובת על 70 מעלות צלזיוס במשך 3 דקות. </li><li style=";text-align:right;direction:rtl"> הסר את המדגם מגוש החימום ולתת לו לעמוד בטמפרטורת חדר למשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 15,000 XG במשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> מוציאים בזהירות את supernatant, משאיר מאחוריו כ 50 μL. </li><li style=";text-align:right;direction:rtl"> הוספת 500 μL של פתרון לשטוף מחדש להשעות את הגלולה ידי pipetting. </li><li style=";text-align:right;direction:rtl"> פיפטה ההשעיה לתוך מערכת צינור ספין מסנן / אוסף. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 15,000 XG במשך 2 דקות. הסר את עמודת צינור איסוף וזורק את הזרימה דרך. החזר את עמודת צינור האיסוף. </li><li style=";text-align:right;direction:rtl"> פיפטה 500 μL של פתרון לשטוף על מסנן הספין. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 15,000 x גרם במשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> מעביר את מסנן הספין לתוך צינור איסוף טרי. </li><li style=";text-align:right;direction:rtl"> פיפטה 50 μL של פתרון elution מחומםעד 70 ° C על גבי במרכז מסנן הספין. </li><li style=";text-align:right;direction:rtl"> דגירה של 2-5 דק &#39;ב 70 ° C. צנטריפוגה ב 15,000 XG דקות 1. </li><li style=";text-align:right;direction:rtl"> פיפטה נוסף 50 μL של פתרון elution מחומם ל -70 מעלות צלזיוס על גבי במרכז מסנן הספין. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 1.2.2.1.18. </li></ol></li><li style=";text-align:right;direction:rtl"> הוספת 100 מיקרוגרם / מ&quot;ל ​​גליקוגן, 0.1 נפח של 3 M חיץ נתרן אצטט (5.2 pH), ו 2.5 כרכים של אתנול אבסולוטי. משקע הלילה ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 15,000 XG במשך 25 דקות ב 4 °. בטל supernatant. </li><li style=";text-align:right;direction:rtl"> שטפו את הכדור עם 1 מ&quot;ל של אתנול 75%. צנטריפוגה ב 15,000 XG במשך 15 דקות ב 4 ° C.. מוציאים בזהירות את אתנול. </li><li style=";text-align:right;direction:rtl"> יבש באוויר במשך 3-5 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף 10 μL של מי nuclease ללא לפזר את הגלולה. </li><li style=";text-align:right;direction:rtl"> חנות ב -70 ° C. </li></ol></li></ol></li><li style=";text-align:right;direction:rtl"> הכשרת RNA וכימות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות ספקטרופוטומטר, לקבוע את ריכוז RNA על ידי NotIng את הספיגה ב 260 ננומטר ו -280 ננומטר. יחס 260/280 צריך להיות 1.8-2.2. </li><li style=";text-align:right;direction:rtl"> בדוק את איכות המדגם RNA באמצעות 0.8% ג&#39;ל agarose אלקטרופורזה 25. הערה: רנ&quot;א הכל ללא פגע איקריוטיים צריך להראות 28S אינטנסיבי 18S rRNA להקות. יחס 28S לעומת עוצמת להקת 18S rRNA עבור הכנת RNA טובה הוא ~ 2. </li></ol></li></ol> 2. ג&#39;ל אלקטרופורזה והסעה הערה: הפרוטוקולים כן חיץ ניתנים בטבלת 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת ג&#39;ל פורמלדהיד (1%) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לשטוף את כל הציוד אלקטרופורזה עם diethylpyrocarbonate (DEPC) מים. </li><li style=";text-align:right;direction:rtl"> ממיסים 2.5 גרם של agarose ב 215 מ&quot;ל מים DEPC לחלוטין במיקרוגל. </li><li style=";text-align:right;direction:rtl"> הוסף 12.5 מ&quot;ל של 10x 3- (N- morpholino) חומצה propanesulfonic (מגבים) חיץ 22.5 מ&quot;ל של פורמלדהיד 37%. </li><li style=";text-align:right;direction:rtl"> יוצקים את התערובת לתבנית המנגנון אלקטרופורזה עם מסרק סמיך 1.5 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> לחסל בועות או לדחוף tשולי עד הקצוות הג&#39;ל במסרק נקי. </li><li style=";text-align:right;direction:rtl"> אפשר הג&#39;ל לחזק בטמפרטורת החדר. </li></ol></li><li style=";text-align:right;direction:rtl"> לדוגמא הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבבים 1-10 מיקרוגרם של RNA סך 11.3 μL של חיץ מדגם. </li><li style=";text-align:right;direction:rtl"> מערבבים 2 מיקרוגרם של הסמן RNA (1 מיקרוגרם / μL) עם 11.3 μL של חיץ מדגם. </li><li style=";text-align:right;direction:rtl"> מוסיפים מים nuclease ללא דגימות 2.2.1 ו 2.2.2 כדי בהיקף כולל של 16 μL. </li><li style=";text-align:right;direction:rtl"> מחממים על 60 מעלות צלזיוס למשך 5 דקות, ולאחר מכן לצנן על הקרח. </li><li style=";text-align:right;direction:rtl"> מערבבים 16 μL של המדגם מ 2.2.3 עם 4 μL של צבע מעקב, המכיל 0.1 מיקרוגרם / μL ethidium ברומיד על הקרח. זהירות: ברומיד Ethidium הוא אולי טרטוגניות ורעילים אם נשאף. קרא גליון בטיחות ברומיד ethidium. </li></ol></li><li style=";text-align:right;direction:rtl"> אלקטרופורזה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 200 מ&quot;ל של 10x מגבים חיץ ל -1,800 מ&quot;ל של DDH 2 O לבצע למאגר פועל מגבים 1x. </li><li style=";text-align:right;direction:rtl"> השתמש למאגר פועל מגבי 1x לשטוף את הבארות של ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> t טרום ריצההוא הג&#39;ל על 20 V למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> טען את דגימות רנ&quot;א לתוך הבארות של ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> מכסים בנייר כסף, כדי למנוע חשיפה לאור. הפעלה ב 35 V עבור כ -17 שעות (לילה) </li><li style=";text-align:right;direction:rtl"> בדוק ולצלם את הג&#39;ל תחת אור UV. </li></ol></li><li style=";text-align:right;direction:rtl"> לְהַעֲבִיר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את ג&#39;ל להסרת החלק בשימוש. </li><li style=";text-align:right;direction:rtl"> הכן 500 מ&quot;ל של 10x חיץ SSC (250 מ&quot;ל של חיץ 20x SSC ו 250 מ&quot;ל מים DEPC). </li><li style=";text-align:right;direction:rtl"> שטפו את הג&#39;ל פעמיים עם חיץ 10x SSC במשך 20 דקות עם רועד ב 50 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> חותכי 1 נייר סינון גיליון גדול מספיק כדי לשמש נייר פתילה, אשר סופג חיץ העברה ממגש ההעברה (איור 1). </li><li style=";text-align:right;direction:rtl"> חותכים 4 חתיכות של נייר סינון לאותו גודל כמו ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> חותכים 1 גיליון קרום ניילון חיובי טעונים לאותו גודל כמו ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> סמן חריץ על הג&#39;ל ואת הקרום כסמן כדי להבטיח את הכיוון הנכון. </li><li style=";text-align:right;direction:rtl"> משרים את הממברנה חיץ 2x SSC במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> יוצקים 500 מ&quot;ל of 10x SSC חיץ למגש ההעברה. </li><li style=";text-align:right;direction:rtl"> הנח את גיליון נייר סינון גדול על פני הצלחת זכוכית להרטיב את נייר הסינון עם חיץ העברה. </li><li style=";text-align:right;direction:rtl"> מרדד כל בועות עם טפטפת. </li><li style=";text-align:right;direction:rtl"> משרי 2 חתיכות של נייר סינון מראש לחתוך חיץ העברה ומניח אותם על המרכז של נייר הסינון משלב 2.4.5. הסר את כל בועות. </li><li style=";text-align:right;direction:rtl"> הנח את הדף בצד ג&#39;ל למטה על נייר הסינון. </li><li style=";text-align:right;direction:rtl"> הנח את קרום על גבי הג&#39;ל. ישר את החריצים של הג&#39;ל ואת הממברנה. </li><li style=";text-align:right;direction:rtl"> מניחים 2 חתיכות של נייר סינון מראש לחתוך על גבי הממברנה ואת להרטיב את נייר הסינון עם חיץ העברה. </li><li style=";text-align:right;direction:rtl"> הסר את כל בועות בין השכבות. </li><li style=";text-align:right;direction:rtl"> החל בניילון סביב הג&#39;ל על מנת להבטיח כי רק מגבה לספוג חיץ עובר דרך ג&#39;ל הממברנה. </li><li style=";text-align:right;direction:rtl"> סטאק את מגבות נייר על גבי נייר הסינון. </li><li style=";text-align:right;direction:rtl"> מניח צלחת זכוכית גדולה על גבי המגבות. </li><li style=";text-align:right;direction:rtl"> מניחים משקל על החלק העליון של הערימה. </li><li style=";text-align:right;direction:rtl">שמור לילה לאפשר RNA להעביר מן הג&#39;ל על הממברנה. </li></ol></li></ol> 3. איתור של N 6 -methyladenosine <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממברנה Crosslinking <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שמור את קרום לחת חיץ 2x SSC. </li><li style=";text-align:right;direction:rtl"> מניחים 2 גיליונות נייר מסנן שקוע עם 10x חיץ SSC לתוך crosslinker UV. </li><li style=";text-align:right;direction:rtl"> מניחים את קרום על גבי נייר הסינון, כשהצד RNA-adsorbed פונה כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> בחר &quot;מצב autocrosslink&quot; (120,000 μJ) ולחץ על כפתור &quot;התחל&quot; כדי להתחיל את ההקרנה. </li><li style=";text-align:right;direction:rtl"> בדוק את הממברנה ואת ג&#39;ל עם לוכד תמונה ג&#39;ל UV כדי לאשר את העברת RNA מן הג&#39;ל על הממברנה. </li></ol></li><li style=";text-align:right;direction:rtl"> immunoblotting <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חסום את הממברנה ב 5% חלב דל שומן מלוחים טריס שנאגרו עם 20 Tween (TBST) חיץ בטמפרטורת החדר למשך שעה 1. </li><li style=";text-align:right;direction:rtl"> לשטוף שלוש פעמים עם חיץ TBST במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה לילה מ 6 (&lt;em&gt; N 6 פתרון נוגדנים -methyladenosine) (1: 1,000 ב 5% דל שומן חיץ חלב TBST) ב 4 ° C.. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה שלוש פעמים עם חיץ TBST במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה ב HRP-מצומדות חמור פתרון נוגדנים נגד ארנב (1: חיץ 2,000 5% דל שומן חלב TBST) במשך שעה 1 בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> לשטוף את הממברנה שלוש פעמים עם חיץ TBST במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> החל את המצע chemiluminescent משופרת (0.125 מ&quot;ל לס&quot;מ 2 של קרום). </li><li style=";text-align:right;direction:rtl"> ללכוד את chemiluminescence עם ההגדרות האופטימליות של תרמי הדיגיטלי, על פי הוראות יצרן 26. </li></ol></li><li style=";text-align:right;direction:rtl"> מ 6 כימות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> למדוד את עוצמת chemiluminescent היחסית מ 6 באמצעות תוכנת ImageJ. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בתפריט התוכנה ImageJ, בחר באפשרות &quot;קובץ&quot; כדי לפתוח את הקובץ הרלוונטי. </li><li style=";text-align:right;direction:rtl"> בחר באפשרות &quot;מלבן&quot; מ ImageJ ולצייר על ידי מסגרת סביבאת האותות. </li><li style=";text-align:right;direction:rtl"> אם RNA ריבוזומלי הוא לא המוקד של ניסויים, להימנע להקות ריבוזומלי 18S ו -28 RNA מ 6 לחישוב. </li><li style=";text-align:right;direction:rtl"> לחץ על &quot;פיקוד&quot; ו &quot;1&quot; כדי לאשר את השביל שנבחר. </li><li style=";text-align:right;direction:rtl"> לחץ &quot;פיקוד&quot; ו &quot;3&quot; כדי להראות את העלילה שנבחרה. </li><li style=";text-align:right;direction:rtl"> לחץ על הכלי &quot;ישר&quot; לצייר קווים כדי להפריד את האזור המפולח. </li><li style=";text-align:right;direction:rtl"> לחץ על הכלי &quot;שרביט&quot; כדי לרשום את המידות. </li><li style=";text-align:right;direction:rtl"> לייצא את הנתונים. </li><li style=";text-align:right;direction:rtl"> לנרמל את רמות א מ 6 עם להקת RNA ריבוזומלי 18S מן המכתים ברומיד ethidium. </li></ol></li></ol></li></ol>

<ol>
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החוקרים אין לי מה לחשוף.

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNaseZap solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9782</td>
			<td style="width:167px;">Protocol 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TRI Reagent solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9738</td>
			<td>Protocol 1.2.1.1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Bromo-3-chloropropane (BCP)</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">B9673</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:96px;">JTbaker</td>
			<td style="width:119px;">8006</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">I9516</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenElute mRNA Miniprep Kit</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">MRN70</td>
			<td>Protocol 1.2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9510</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:96px;">Fluka</td>
			<td style="width:119px;">71183</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease-free water</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9930</td>
			<td>Protocol 1.2.2.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D5758</td>
			<td>Protocol 2.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">A426</td>
			<td>Protocol 2.1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">M1254</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37% formaldehyde Solution</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F8775</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA, Disodium Salt</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">8993</td>
			<td>Protocol 2.1.3 10X MOPS buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">formamide</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F7503</td>
			<td>Protocol 2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA Millennium Marker</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM7150</td>
			<td>Protocol 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethidium Bromide</td>
			<td style="width:96px;">Amresco</td>
			<td style="width:119px;">X328</td>
			<td>Protocol 2.2.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ficoll 400</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">17-0300-10</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromophenol blue</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">114391</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene Cyanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">X4126</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">3624</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisodium citrate</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">S1804</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">filter paper</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN6101M</td>
			<td>Protocol 2.4.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GE Hybond-N+ membrane</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN303B</td>
			<td>Protocol 2.4.6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stratalinker UV Crosslinker 2400</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">400075</td>
			<td>Protocol 3.1.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gel Catcher 1500</td>
			<td style="width:96px;">ANT Technology</td>
			<td style="width:119px;">Gel Catcher 1500</td>
			<td>Protocol 3.1.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-m6A (N6-methyladenosine)</td>
			<td style="width:96px;">Synaptic Systems</td>
			<td style="width:119px;">202003</td>
			<td>Protocol 3.2.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934</td>
			<td>Protocol 3.2.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc MP System</td>
			<td style="width:96px;">BIO-RAD</td>
			<td style="width:119px;">1708280</td>
			<td>Protocol 3.2.8</td>
		</tr>
	</tbody>
</table>

a method for measuring rna n6-methyladenosine modifications in cells and tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

לאחר 14 ימים בשלב יממה רגיל-כהה אור, עכברי wild-type הונחו בחשכה מתמדת. RNAs מהכבד נדגמו ב 4 במרווחים hr ולמד עם סופג הצפוני שונה. מתילציה של rRNAs, mRNAs, ו RNA הקטן התגלתה בבירור (איור 2). ההשוואה בין זמני יממה שונים (CT) ניתן לחשב במדויק עם תקן 18S rRNA. הייתה תנודת יממה חזקה של רמות מ 6 ב rRNA, mRNA, ו RNA הקטן. כדי למנוע את ההפרעה שהוצעה על ידי rRNA, RNAs polyadenylated יכול להיות מטוהר, כמו בשלב 1.2.2. לאחר הטיהור, rRNA ניתן למנוע במידה רבה, כדי לאפשר ויזואליזציה טובה יותר של RNAs האחר (איור 3). <img alt="איור 1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html> g&gt; איור 1: רכבת יחידת העברת כתם. יחידת העברת נימים עבור סופג את הרנ&quot;א על ​​הקרום מוצגת. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/54672/54672fig2.jpg" /> איור 2: קצב היממה של בחינות הבגרות מ 6 ב C57BL / 6J. נתון זה מראה את מ 6 כתם של RNA הכולל כבדים עכברי-בר הקריב ביום הראשון של השלב הכהה כהה לאחר 14 ימים של שלב אור כהה רגיל ב 4 במרווחי h. כימות נעשה באמצעות מ 6 הפרש צפיפות התמונה בין 18S ו -28 rRNA. מ &#39;6 שפע היה מנורמל הלהקה 18S rRNA מן הג&#39;ל פורמלדהיד בתחתית.et = &quot;_ blank&quot;&gt; לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו. <img alt="איור 3" src="/files/ftp_upload/54672/54672fig3.jpg" /> איור 3: כתמי הקלע זרק 6 עם או בלי rRNA. כתמי נציג מ 6 א &#39;של רנ&quot;א הכל ו- RNA polyadenylated מן הכבדים של עכברי-בר מוצגים. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig3large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" style=";text-align:right;direction:rtl"><tbody><tr><td> 1 </td><td colspan="3"> חיץ 10x מגבים (pH 7.0, להגן מפני אור) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> מגבים </td><td> 41.85 </td><td> g </td><td> 0.2 M </td></tr><tr><td></td><td colspan="3"> אצטט נתרן </td><td> 4.1 </td><td> g </td><td>0.05 מ &#39; </td></tr><tr><td></td><td colspan="3"> EDTA, Disodium מלח </td><td> 3.7 </td><td> g </td><td> 0.01 M </td></tr><tr><td></td><td colspan="3"> מי DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> סה&quot;כ </td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3"> מערבבים בטמפרטורת החדר </td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20x חיץ SSC (pH 7.0) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> נתרן כלורי </td><td> 175.3 </td><td> g </td><td> 3 M </td></tr><tr><td></td><td colspan="3"> ציטראט Trisodium </td><td> 88.3 </td><td> g </td><td> 0.3 M </td></tr><tr><td></td><td colspan="3"> מי DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> סה&quot;כ </td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3"> מערבבים בטמפרטורת החדר, ואז החיטוי </td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3"> דיי מעקב </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> חיץ 10x מגבים </td><td> 500 </td><td> μL </td><td> 1x </td></tr><tr><td></td><td colspan="3"> Ficoll 400 </td><td> 0.75 </td><td> g </td><td> 15% </td></tr><tr><td></td><td colspan="3"> bromophenol כחול </td><td> 0.01 </td><td> g </td><td> 0.2% </td></tr><tr><td></td><td colspan="3"> קסילן Cyanol </td><td> 0.01 </td><td> g </td><td> 0.2% </td></tr><tr><td></td><td colspan="3"> מי DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> סה&quot;כ </td><td> 5 </td><td> מ&quot;ל </td><td></td></tr><tr><td></td><td colspan="3"> חנות ב -20 ° C </td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> מי DEPC </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> לדלל יחס של 1: 1,000 של DEPC DDH 2 O </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> מערבבים למשך הלילה בטמפרטורת החדר, ואז החיטוי </td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3"> חיץ מדגם (להגן מפני אור) </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> חיץ 10x מגבים </td><td> 200 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 37% פורמלדהיד </td><td> 270 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> Formamide </td><td> 660 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> חנות ב -20 ° C </td><td></td><td></td><td></td></tr></tbody&gt; </table> טבלה 1: חוצצים ופתרונות.

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

שיטה סופגת צפונית שונה למדידת N 6 -methyladenosine (מ 6 א) שינויים ב RNA מתוארת. השיטה הנוכחית יכולה לזהות שינויים ב RNAs ובקרות מגוונות תחת עיצובים ניסיוניים שונים.

שיטה למדידת RNA<em&gt; N</em<sup&gt; 6</sup&gt; שינויי -methyladenosine ב תאים ורקמות

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

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Nanyang Technological University

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JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

10.8K Views.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  שיטה סופגת צפונית שונה למדידת N 6 -methyladenosine (מ 6 א) שינויים ב RNA מתוארת. השיטה הנוכחית יכולה לזהות שינויים ב RNAs ובקרות מגוונות תחת עיצובים ניסיוניים שונים. 

Video: A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

שיטה יעילה עבור הבידוד של רנ&quot;א מאוד מטוהר מזרעים לשימוש ב כמוני transcriptome ניתוח

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

שיטה למדידת מטבוליזם ב ממוינות תת-אוכלוסיות של קהילות Cell מורכבות באמצעות איתור איזוטופ יציב

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

בידוד של חלבונים ביניים חלבונים מ רקמות עכבר מרובות לחקר ההזדקנות הקשורים שינויים טרנסלוציוניים

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

הכנת הדוגמא לזיהוי מבוססי ספקטרומטר מסה של RNA-איגוד אזורים

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

שיטה ערכי צבע מוחלטים למדידת תכולת ברזל צמחים

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

ניתוח Toeprinting של תרגום חניכה היווצרות מורכבות על יונקים mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

Microgravimetry dissipative ללמוד את הדינמיקה איגוד של פוספוליפיד איגוד חלבון Annexin A2 כדי Bilayers השומנים הנתמכות על-ידי מוצק באמצעות מהוד קוורץ

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

מדידת ופרשנות חמצן קצב הצריכה כולה לטוס הראש מקטעים

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

הדמיית פלורסנט של מנגו-מתויג RNA ב-Pvc פוליאקרילאמיד באמצעות שיטת Poststaining

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

תרגום הריבוכמה אהדה לטיהור (מלכודת) לבידוד רנ א מתאי האנדותל בvivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...