विकासशील और परिपक्व रेटिना न्यूरॉन्स की रूपरेखा एकल कक्ष
Summary
एक रेटिना की कोशिकाओं और उनके cDNAs के बाद प्रवर्धन के अलगाव के लिए एक विधि का वर्णन है. एकल सेल transcriptomics एक ऊतक में सेलुलर विजातिता के वर्तमान की डिग्री से पता चलता है और दुर्लभ सेल आबादी के लिए नया मार्कर जीन uncovers. साथ प्रोटोकॉल के लिए कई अलग अलग प्रकार के सेल के अनुरूप समायोजित किया जा सकता है.
Abstract
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.
Protocol
Discussion
अध्ययन की एक कभी विस्तार की संख्या आबादी है कि उनके जीन अभिव्यक्ति 6,8 के संबंध में अधिक सजातीय होना माना गया में मजबूत परिवर्तनशीलता सेल के लिए सेल का खुलासा कर रहे हैं. कम से कम एक उदाहरण में, इस जीन ?…
Disclosures
The authors have nothing to disclose.
Materials
Reaction mixtures:
Cell Lysis buffer
| 0.45 μ | 10X reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2) |
| 0.23 μl | 10% NP-40 |
| 0.23 μl | 0.1M DTT |
| 0.05 μl | RNase Inhibitor (40 U/μl) |
| 0.05 μl | SUPERase-In (20U/μl) |
| 0.13 μl | Modified Oligo d(T) primer (TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT) |
| 0.09 μl | dNTPs (2.5 mM each) |
| 3.27 μl | dH20 (use molecular biology grade for all reaction mixtures) |
Tailing Reaction Mixture
| 0.18 μl | 100 mM dATP |
| 0.6 μl | 10X reaction buffer(100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2) |
| 4.62 μl | dH2O |
| 0.3 μl | TdT (400 U/μl) |
| 0.3 μl | RNase H (2 U/μl) |
PCR Reaction Mixture
| 10 μl | 10x Ex-Taq HS Buffer with Mg2+ |
| 10 μl | 2.5 mM dNTPs |
| 0.2 μl | Modified Oligo d(T) primer (10 μg/μl) |
| 1 μl | Ex-Taq HS Polymerase (5U/μl) |
| 68.8 μl | dH2O |
10X One-Phor All Buffer
| 0.5M | Potassium acetate |
| 0.1M | Tris acetate (pH 7.6) |
| 0.1M | Magnesium acetate |
Solutions:
10X Phosphate buffered saline (1 liter)
80g NaCl
2g KCl
14.4 g Na2PO4
2.4 g KH2PO4
adjust to pH 7.4 with NaOH and bring the volume to 1 liter with water
| Name of the reagent | Company | Catalog number | Comments |
| Vertical needle puller | David Kopf Instruments | Model 750 | |
| Microcapillary tubes | Sigma | P0674 | |
| Aspirator tube assembly | Sigma | A5177 | |
| Corning filter tips (100-1000 μl | Fisher Scientific | 07-200-265 | |
| Hank’s balanced salt solution (1X) | Lonza BioWhittaker | 10-508F | |
| HEPES buffer (1M) | MP Biomedicals | 091688449 | |
| Bovine serum albumin | Sigma | A9418 | |
| DNase I | Roche | 04716728001 | |
| papain | Worthington | LS03126 | |
| Superscript III | Invitrogen | 18080-044 | |
| RNase Inhibitor | Applied Biosystems | AM2682 | These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear. |
| SUPERase-In | Applied Biosystems | AM2694 | These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear. |
| Modified Oligo d(T) primer (gel purified) | Oligos ETC | We have used primers purchased from many different companies and have seen the most reliable and reproducible results from Oligos ETC. | |
| T4 gene 32 protein | New England Biolabs | M0300L | |
| Exonuclease I | New England Biolabs | M0293S | |
| TdT | Roche | 3 333 574 | As with other reagents, using a TdT enzyme from other companies gave more variable results. |
| RNase H | Invitrogen | 18021-014 | |
| Ex-Taq HS Polymerase | Takara | RR006A | |
| Biotin N6-ddATP | Enzo Biosciences | 42809 |
Table 1. Specific reagents and equipment.
References
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