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

ABSTRACT:

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

SUBMITTER: Tavares RL 

PROVIDER: S-EPMC3454941 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature