A specific and quick gene expression study in mouse ES cells.
OBJECTIVE
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.
Tavares
Rubens L C
RL
Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. kpxu@med.cornell.edu.
Xu
Kangpu
K
Zhang
Chenyang
C
de Freitas
Vilmon
V
D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger