Saltar al contenido
Merck

Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system.

Nature protocols (2018-09-16)
Evelina Tutucci, Maria Vera, Robert H Singer
RESUMEN

The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
Fosfato de potasio monobasic, ACS reagent, ≥99.0%
Sigma-Aldrich
Poli-L-lisina solution, 0.1 % (w/v) in H2O
Sigma-Aldrich
D-(+)-Glucosa, ≥99.5% (GC)
Sigma-Aldrich
Base Trizma®, BioPerformance Certified, meets EP, USP testing specifications, suitable for cell culture, ≥99.9% (titration)
Sigma-Aldrich
2- Mercaptoetanol, ≥99.0%
Sigma-Aldrich
Hidróxido de sodio, BioXtra, ≥98% (acidimetric), pellets (anhydrous)
Sigma-Aldrich
Ácido desoxirribonucleico sodium salt from salmon testes
Sigma-Aldrich
Ribonucleasa A from bovine pancreas, Type I-A, powder, ≥60% RNase A basis (SDS-PAGE), ≥50 Kunitz units/mg protein
Sigma-Aldrich
Lithium acetate dihydrate, reagent grade
Sigma-Aldrich
Deoxyribonucleic acid, single stranded from salmon testes, For hybridization
Sigma-Aldrich
DAPI, dilactate, ≥98% (HPLC)