ברצוננו להודות V. קאפור ב Core cytometry זרימת CCR ETIB עבור לעזרתה בביצוע מיון התא במהלך הפיתוח של פרוטוקול זה. אנו מודים גם מ Raffeld ויחידת האבחון המולקולרי CCR LP וג &#39;ג&#39; ו ואת רצף NHLBI דנ&quot;א ג&#39;נומיקס Core עבור לעזרתם בביצוע qPCR במהלך הפיתוח של פרוטוקול זה. מחקר זה נתמך על ידי התכנית העירונית של NIH.

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The authors have nothing to disclose.

הצגנו שיטה לבידוד בתאי יונקים בודדים מתוך אוכלוסייה של תאי חסיד הגדלים בתרבית ועבור מנסה לאמוד את הביטוי של כ 96 גנים בכל תא. הכנה מראש טוב הוא קריטי עבור בשיטה זו כדי לעבוד היטב. בפרט, בעיצוב פריימר בדיקות זוגות מסוימים אל תעתיקי עניין (שלבי 1.2-1.3) צורכים זמן אבל חשובי צ?...

תאים בודדים בתוך אוכלוסייה יכול להראות תגובות ומעולם שונות לגירוי 1 פיסיולוגי אחיד, 2, 3, 4. הווריאציה הגנטית של תאים באוכלוסייה היא מנגנון אחד עבור מגוון זה של תגובות, אבל יש גם כמה גורמים שאינם גנטיים שיכול להגדיל את ההשתנות של תגובות, גם באוכלוסייה משובטת של תאים. לדוגמא, הרמות של חלבונים בודדים מולקולות איתות חשובות אחרות יכולות להשתנות על בסיס תא אחר תא, והולידו וריאצית פרופילי ביטוי גנים במורד זרם. בנוסף, הפעלת גנים יכולה להתרחש התפרצויות קצר משך התמלילים 5, 6 שעשויות להיות מוגבל למספר קטן יחסית של תמלילים לכל פרץ 7, 8, 9. כגוןstochasticity ב הפעלת הגן יכול לתרום השתנות מאוד תגובות ביולוגיות והוא יכול לספק יתרון סלקטיבי מיקרואורגניזמים 10 ו בתאי יונקים 1, 2 מגיבים על גירוי פיזיולוגי. בשל שני המקורות הגנטיים שאינם גנטיים של וריאציה, פרופיל ביטוי גנים של כל תא נתון בתגובה לגירוי עשוי להיות שונה באופן משמעותי מפרופיל ביטוי גנים הממוצע המתקבל מדידת התגובה בתפזורת. קביעת המידה שבה תאים בודדים מראים השתנות בתגובה לגירוי דורשת טכניקות עבור הבידוד של תאים בודדים, מדידת רמות ביטוי תמלילי העניין, ועל הניתוח חישובית של נתוני הביטוי שהתקבלו. ישנן מספר גישות מנסים לאמוד ביטוי גנים בתאים יחידים, מכסי מגוון רחב של עלויות, מספר תמלילי נחקר, ודיוק של כימות. לדוגמא, תא בודד RNA-seq מציע עומק רחב של כיסויי תמליל ואת היכולת לכמת אלף תמלילים ברורים עבור הגנים הביעו הגבוהה ביותר בתאים בודדים; עם זאת, העלות הכרוכה עומק רצף כזה עלולה להיות מרתיעה, אם כי עלויות ממשיכות לרדת. לעומת זאת, קרינת RNA מולקולה בודדת הכלאה באתרו (FISH smRNA) מציעה כימותים מדויקים של תמלילים אפילו נמוך לבטא גנים במחיר סביר לכל גן של עניין; עם זאת, רק מספר קטן של גני מטרה ניתן assayed בתא נתון על ידי גישה זו. מבחני PCR מבוססים כמוני, כמתואר בפרוטוקול זה, לספק את שביל זהב בין הטכניקות הללו. מבחנים אלו להעסיק המכונים PCR microfluidic בזמן אמת לכמת עד 96 תמלילי עניין בכל פעם עד 96 תאים. בעוד כל אחת מהשיטות הנ&quot;ל יש עלויות חומרה נדרשות, את העלות של כל assay qPCR פרט היא יחסיתנָמוּך. פרוטוקול זה מותאם מאחד שהוצע על ידי היצרן של מכונת PCR microfluidic בזמן אמת (פרוטוקול ADP 41, Fluidigm). כדי להפעיל את הערכת מספרם המוחלט של כל תמליל בגישה מבוססת PCR, הרחבנו את הפרוטוקול לעשות שימוש לגבי הבקרה פנימית על amplicons גן המטרה המוכן שניתן להשתמש בה בכל ניסויים מרובים. כדוגמה של טכניקה זו, כימות של ביטוי גנים מוסדר על ידי p53 מדכא גידולים בתאי סרטן שד אנושיים MCF-7 מתוארת 11. התאים מאותגרים עם סוכן כימי שגורם הפסקות פעמים גדיל DNA. מחקרים קודמים הראו כי התגובה p53 הפסקות DNA פעמיים גדיל מפגין מידה רבה של ההטרוגניות בתאים בודדים, הן מבחינת רמות ה- p53 12 ו בהפעלת גני מטרה מובחנת 11. יתר על כן, p53 מסדיר את הביטוי של למעלה מ -100היטב מאופיין גני מטרה המעורבים מסלולים רבים במורד הזרם, כולל עיכוב מחזור התא, אפופטוזיס, והזדקנות 13, 14. מאז התגובה בתיווך p53 בכל תא היא גם מורכבת ומשתנה, הניתוח של יתרונות המערכת מגישה שבה כמעט 100 גני המטרה ניתן נחקר בו זמנית בתאים בודדים, כמו זו מתוארת להלן. עם שינויים קלים (כגון שיטות חלופיות עבור בידוד תא בודד תמוגה), הפרוטוקול ניתן להתאים בקלות ללמוד מגוון רחב של סוגי תאים של יונקים, תעתיקים, ואת תגובות הסלולר. עם הכנה מראש נכונה, סבב מיון תא ומדידת ביטוי גנים יכול להתנהל על פי פרוטוקול זה על פני תקופה של שלושה ימים. העיתוי הבא הוא הציע: מראש, בחר את תמלילי העניין, לזהות ולאמת את הזוגות פריימר כי להגביר את cDNA מאלה transcripts, ולהכין את הסטנדרטים ותערובות פריימר באמצעות פריימרים אלו. ביום 1, לאחר טיפול בתא, קציר ולמיין את התאים, לבצע שעתוק לאחור והגברת יעד ספציפי, ולטפל דגימות עם exonuclease להסיר פריימרים מאוגדים. ביום 2, לבצע בקרת איכות על תאים ממוינים באמצעות qPCR. לבסוף, ביום 3, למדוד את ביטוי הגנים בתאים המסודרים באמצעות microfluidic qPCR. איור 1 מסכם את השלבים הכרוכים.

<table><tbody><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74134</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td><td>ThermoFisher</td><td>4374966</td><td></td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>New England BioLabs</td><td>M0530S</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>Quant-iT High-Sensitivity dsDNA Assay Kit</td><td>ThermoFisher</td><td>Q33120&amp;nbsp;</td><td></td></tr><tr><td>2.0 ml low adhesion microcentrifuge tubes</td><td>USA Scientific</td><td>1420-2600</td><td></td></tr><tr><td>DNA Suspension Buffer</td><td>Teknova</td><td>T0221</td><td></td></tr><tr><td>Axygen 0.2 ml Maxymum Recovery Thin Wall PCR Tubes</td><td>Corning</td><td>PCR-02-L-C</td><td></td></tr><tr><td>GE 96.96 Dynamic Array DNA Binding Dye Sample &amp;amp; Assay Loading Reagent Kit</td><td>Fluidigm</td><td>100-3415</td><td></td></tr><tr><td>HyClone RPMI 1640 media</td><td>GE Healthcare Life Sciences</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Bovine Serum, Certified (US)</td><td>ThermoFisher</td><td>16000-044</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution</td><td>Corning</td><td>30-004-CI</td><td></td></tr><tr><td>Neocarzinostatin</td><td>Sigma</td><td>N9162</td><td></td></tr><tr><td>ELIMINase</td><td>Decon Labs</td><td>1101</td><td></td></tr><tr><td>SUPERase-In</td><td>ThermoFisher</td><td>AM2696</td><td></td></tr><tr><td>CellsDirect One-Step qRT-PCR Kit</td><td>ThermoFisher</td><td>11753500</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DNA</td><td>Affymetrix</td><td>14380 10 MG</td><td></td></tr><tr><td>ThermalSeal Sealing Film, Sterile</td><td>Excel Scientific</td><td>STR-THER-PLT</td><td></td></tr><tr><td>BD FACSAria IIu</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>HyClone Trypsin 0.05%</td><td>GE Healthcare Life Sciences</td><td>SH30236.01</td><td></td></tr><tr><td>PBS, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Falcon 40 &amp;micro;m Cell Strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Exonuclease I</td><td>New England BioLabs</td><td>M0293S</td><td></td></tr><tr><td>SsoFast EvaGreen Supermix with Low ROX</td><td>Bio-Rad</td><td>172-5210</td><td></td></tr><tr><td>96.96 Dynamic Array IFC for Gene Expression (microfluidic qPCR chip)</td><td>Fluidigm</td><td>BMK-M-96.96</td><td></td></tr><tr><td>IFC Controller HX (loading machine)</td><td>Fluidigm</td><td></td><td></td></tr><tr><td>BioMark or BioMark HD (microfluidic qPCR machine)</td><td>Fluidigm</td><td></td><td></td></tr><tr><td>Real-Time PCR Analysis software&amp;nbsp;</td><td>Fluidigm</td><td></td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks</td><td></td><td></td></tr></tbody></table>

single-cell gene expression profiling using facs and qpcr with internal standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

סקירה כללית של הפרוטוקול מוצגת באיור 1, כוללת צעדים לטיפול תא, הבידוד של תאים בודדים על ידי FACS, דור מראש הגברה של ספריות cDNA מ lysates תא בודד, האישור של ספריות cDNA מתא בודד ב בארות מיון, וכן מדידה של ביטוי גנים על ידי qPCR. <p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards at JoVE.com

פרופיל ביטוי גנים חד-תא באמצעות FACS ו-qPCR עם תקנים פנימיים | גנטיקה | וִידֵאוֹ

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

תאים יחידים Gene Expression Profiling שימוש FACS ו qPCR עם סטנדרטים פנימיים

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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16.3K Views.  National Cancer Institute. We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Video: Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

ביטוי גנים תא בודד באמצעות Multiplex RT-qPCR לאפיון הטרוגניות של אוכלוסיות הלימפה נדירים

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

בידוד ואפיון של תאים בודדים מעוברים דג הזברה

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Developmental Biology

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

הגישה קומבינטורית תא בודד כדי לאפיין את מולקולרית והטרוגניות Immunophenotypic של גזע האדם ואוכלוסיות קדמון

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

מתא בודד פרופיל של נוירונים רשתית מתפתחות מבוגרים

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Neuroscience

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

ניתוח תא יחיד של אללים פעילים בתעתיק על ידי מולקולה אחת FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Biochemistry

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

פרופיל ביטוי גנים של חיידקים הדבקה באמצעות פלטפורמה דיגיטלית ברקוד

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

Immunology and Infection

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

רקמות קרינה הופעלו מיון תאים (FACS) וניתוח ביטוי גנים של נוירונים Fos להביע מ טרי קפוא מוח החולדה

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

פרופיל תא יחיד תעתיק של cardiomyocytes עכבר למבוגרים

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

ניתוח של שעתוק גנים מהתא הבודד על ידי RNA פלורסנט<em&gt; באתר</em&gt; הכלאה (דגים)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

הדמיה וניתוח של מולקולות ה-mRNA באמצעות הקרינה<em&gt; באתרו</em&gt; הכלאה ב<em&gt; Cerevisiae Saccharomyces</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

תאים יחידים Gene Expression Profiling שימוש FACS ו qPCR עם סטנדרטים פנימיים

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