RESUMO

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.

Assuntos

Células-Tronco Pluripotentes , Biossíntese de Proteínas , Humanos , Células-Tronco Pluripotentes/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sacarose/metabolismo