A specific and quick gene expression study in mouse ES cells.

Overview

PURPOSE: To study gene expression at single embryonic stem cell colony levels with a new RT-PCR protocol. METHODS: Forty-five mouse ES cell colonies were retrieved at the 5th, 10th, 15th, 20th and 25th passages. The pluripotent state was analyzed for OCT-4 and Nanog, and beta-actin as a control for the presence of templates. RT-PCR was done using the SuperScript III CellsDirect cDNA Synthesis System. Every 2 or 3 days just before passage, a single colony was loaded into a 0.5 ml PCR tube containing 10 mul of resuspension buffer using a pulled glass pipette. RESULTS: The RT-PCR protocol was completed in less then 150 min. All colonies were positive for OCT-4 and beta-actin and 42 out of 45 were positive for Nanog. CONCLUSIONS: This protocol requires as little as 10 pg of total RNA starting material and is therefore useful for low cell number tissues, such as single stem cell colonies or preimplantation embryonic materials.