Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization.
Nat Protoc
; 8(6): 1100-13, 2013 Jun.
Article
em En
| MEDLINE
| ID: mdl-23680982
ABSTRACT
We present a protocol for measuring the absolute number of mRNA molecules from a gene of interest in individual, chemically fixed Escherichia coli cells. A set of fluorescently labeled oligonucleotide probes is hybridized to the target mRNA, such that each mRNA molecule is decorated by a known number of fluorescent dyes. Cells are then imaged using fluorescence microscopy. The copy number of the target mRNA is estimated from the total intensity of fluorescent foci in the cell, rather than from counting discrete 'spots' as in other currently available protocols. Image analysis is performed using an automated algorithm. The measured mRNA copy number distribution obtained from many individual cells can be used to extract the parameters of stochastic gene activity, namely the frequency and size of transcription bursts from the gene of interest. The experimental procedure takes 2 d, with another 2-3 d typically required for image and data analysis.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
/
Hibridização in Situ Fluorescente
/
Escherichia coli
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos