isolation of brain from drosophila larvae and harvest of s2 schneider cells

כימות אורך הפולי(A) של גנים ממערכת העצבים דרוזופילה

Most eukaryotic mRNAs are posttranscriptionally polyadenylated at their 3&#8242; terminus in the nucleus by the addition of non-templated adenosines by canonical poly(A) polymerases. An intact poly(A) tail is pivotal throughout the lifecycle of mRNA, as it is essential for mRNA nuclear export1, facilitates interaction with poly(A)-binding proteins to enhance translational efficiency2, and imparts resistance against degradation3. In certain cases, the poly(A) tail can also undergo extension in the cytoplasm, facilitated by noncanonical poly(A) polymerases4. In the cytoplasm, poly (A) tail length dynamically changes and influences the life span of the mRNA molecule. Numerous polymerases and deadenylases are known for modulating tail length5,6,7. For example, the shortening of poly(A) tails correlates with translational repression, whereas the lengthening of poly(A) tails enhances translation8,9.
Accumulating genomic studies have demonstrated the fundamental significance of the poly(A) tail length across various facets of eukaryotic biology. This includes roles in germ-cell development, early embryonic development, neuronal synaptic plasticity for learning and memory, and the inflammatory response10. There have been numerous methods and assays developed for measuring poly(A) tail lengths. For example, the RNase H/oligo(dT) assay takes advantage of RNase H in the presence or absence of oligo(dT) to study poly(A) tail length11,12. Other methods to study poly(A) tail include the PCR amplification of 3&#39; ends such as rapid amplification of cDNA ends poly(A) test (RACE-PAT)12,13 and the ligase-mediated poly(A) test (LM-PAT)14. Further modifications of the PAT assay include ePAT15 and sPAT16. Enzymatic G-tailing17,18 or G/I-tailing&#160;of the 3&#39; end are other variations of the PAT assay. Further modification of these techniques includes the use of fluorescently labeled primers along with capillary gel electrophoresis for high-resolution analysis, referred to as the high-resolution poly(A) test (Hire-PAT)19. These PCR-driven assays allow fast and high-sensitivity poly(A) length quantitation.
With the development of next-generation sequencing, a high-throughput sequencing method, such as PAL-seq20 and TAIL-seq21, allows polyadenylation analyses at a transcriptome-wide scale. However, these methods provide only short sequencing reads of 36-51 nucleotides. Therefore, FLAM-Seq22 was developed for global tail length profiling of full-length mRNA and provides long reads. Nanopore technology23 provides PCR-independent, direct RNA, or direct cDNA sequencing for poly(A) tail length estimations. However, these high-throughput methods are not without limitations. They require large amounts of starting materials, are expensive, and time-consuming. Moreover, analyzing rare transcripts can be extremely challenging with high-throughput methods, and low-throughput PCR-based methods still provide an advantage when a small number of transcripts need to be analyzed, for pilot experiments, and validation of other methods.
We have recently demonstrated that Dscam1 mRNAs contain short poly(A) tails in Drosophila, which necessitates a non-canonical binding of the cytoplasmic poly(A)-binding protein on Dscam1 3&#39;UTR using the G/I tailing method24. Here we provide a streamlined procedure for tissue preparation and quantifying poly(A) length of mRNAs from the Drosophila nervous system and Drosophila S2 cells.

מחבר זרקור: לחקור את הגבול של מחקר mRNA עם טכניקות ניתוח זנב Poly A

מדידת אורך זנב Poly A מזחלי דרוזופילה מוח וקו תאים

Polyadenylation is a post-transcription modification. This add poly-A tails to the three-prime end of mRNA molecules. Dysregulation of polyadenylation has been associated with abnormal gene expression and various diseases, including neurological disorders.Our method provides a cost-effective, high-resolution analysis for measuring poly-A lengths. High-throughput methods for poly-A tail analysis, like next generation sequencing, and a high-throughput sequencing provides high-content information. However, analyzing rare transcripts can be extremely challenging with these methods.PCR-based methods are usually low-throughput. However, they can be easily used to analyze a small number of transcripts. The GI-tailing method is one of them.With this method, we discovered that Dscam mRNAs contain short poly-A tails. Dscam is a neuronal cell adhesion molecule. It is involved in many important neurodevelopmental processes.Because Dscam mRNA contains short poly-A tails unlike most mRNAs, Dscam mRNA translation is under unique gene regulation, which is one of the questions we are trying to answer.

בידוד המוח מזחלי דרוזופילה וקציר תאי שניידר S2

כדי להתחיל, הניחו את זחלי הדרוזופילה בצלחת פטרי ריקה בקוטר 35 מ"מ והשתמשו במלקחיים כדי לשטוף אותם פעמיים במי ברז כדי להסיר את שאריות המזון. מניחים 10 זחלים על צלחת דיסקציה המכילה PBS קר כקרח. מקמו את הצד הגבי של הזחל כלפי מעלה והצמידו כל קצה לצלחת.בצע חתך קטן על דופן הגוף של הקצה האחורי. בעזרת מספריים מיקרודיסקציה, חותכים את דופן הגוף לאורך קו האמצע הגבי לכיוון הקצה הקדמי. שטפו את פנים הזחל שלוש פעמים עם PBS באמצעות פיפטה של פסטר כדי לחשוף את המוח.אתרו והרימו את המוח באמצעות מלקחיים, ובודדו את המוח בעזרת מספריים מיקרודיסקציה. העבירו את המוחות המנותחים לצינור מיקרו-צנטריפוגה מלא ב-PBS קר כקרח ואספו את כל המוחות על קרח. בצע מיצוי RNA באמצעות ערכת מיקרופרפ RNA.עבור בידוד תאי שניידר S2, הוסף חמישה מיליליטר של PBS קר כקרח לתרבית התאים של שניידר. מעבירים את התאים לצינור של 15 מיליליטר, ואז צנטריפוגה ב-1000 גרם למשך חמש דקות בארבע מעלות צלזיוס כדי להוציא את התאים. בעזרת פיפטה, שטפו את התאים פעמיים עם PBS קר כקרח וצנטריפוגה שוב.בצע מיצוי RNA באמצעות ערכת RNA miniprep.

isolation-brain-from-drosophila-larvae-harvest-s2-schneider

Research

JoVE Journal

Neuroscience

Isolation of Brain from Drosophila Larvae and Harvest of S2 Schneider Cells

69 Views.  University of Nevada, Reno. כדי להתחיל, הניחו את זחלי הדרוזופילה בצלחת פטרי ריקה בקוטר 35 מ"מ והשתמשו במלקחיים כדי לשטוף אותם פעמיים במי ברז כדי להסיר את שאריות המזון. מניחים 10 זחלים על צלחת דיסקציה המכילה PBS קר כקרח. מקמו את הצד הגבי של הזחל כלפי מעלה והצמידו כל קצה לצלחת.בצע חתך קטן על דופן הגוף של ה?...

Video: בידוד המוח מזחלי דרוזופילה וקציר תאי שניידר S2

Measurement of Poly A Tail Length from Drosophila Larva Brain and Cell Line

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is tightly regulated by cellular processes. Dysregulation of mRNA polyadenylation has been associated with abnormal gene expression and various diseases, including cancer, neurological disorders, and developmental abnormalities. Therefore, comprehending the dynamics of polyadenylation is vital for unraveling the complexities of mRNA processing and posttranscriptional gene regulation. 
This paper presents a method for measuring poly(A) tail lengths in RNA samples isolated from Drosophila larval brains and Drosophila Schneider S2 cells. We employed the guanosine/inosine (G/I) tailing approach, which involves the enzymatic addition of G/I residues at the 3' end of mRNA using yeast poly(A) polymerase. This modification protects the RNA's 3' end from enzymatic degradation. The protected full-length poly(A) tails are then reverse-transcribed using a universal antisense primer. Subsequently, PCR amplification is performed using a gene-specific oligo that targets the gene of interest, along with a universal sequence oligo used for reverse transcription. 
This generates PCR products encompassing the poly(A) tails of the gene of interest. Since polyadenylation is not a uniform modification and results in tails of varying lengths, the PCR products display a range of sizes, leading to a smear pattern on agarose gel. Finally, the PCR products are subjected to high-resolution capillary gel electrophoresis, followed by quantification using the sizes of the poly(A) PCR products and the gene-specific PCR product. This technique offers a straightforward and reliable tool for analyzing poly(A) tail lengths, enabling us to gain deeper insights into the intricate mechanisms governing mRNA regulation.

The protocol describes an efficient and reliable method for quantifying the poly(A) length of the gene of interest from the Drosophila nervous system, which can be easily adapted to tissues or cell types from other species.

Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is ...