title: Applications of quantitative polymerase chain reaction protein assays during reprogramming.
abstract: The capability to reprogram human somatic cells to induced pluripotent stem cells (iPSCs) has opened a new area of biology and provides unprecedented access to patient-specific iPSCs for drug screening, disease models, and transplantation therapies. Although the process of obtaining iPSC lines is technically simple, reprogramming is a slow and inefficient process consisting of a largely uncharacterized chain of molecular events. To date, researchers have reported a wide range of reprogramming efficiencies, from <0.01% to >1%, depending on the specific reprogramming factors used, the mode of delivery of the reprogramming factors, properties of the starting cells, and culture conditions. We have applied a quantitative polymerase chain reaction methodology, TaqMan Protein Assays to directly quantify the kinetics, and cellular levels of crucial transcription factors during the reprogramming process. Further, we have used the assays to ascertain the threshold levels of reprogramming protein factors required to generate iPSC colonies, to characterize the protein expression signatures of different iPSC lines, and to rapidly identify iPS versus non-iPSC colonies based on expression of pluripotency markers. These data demonstrate that TaqMan Protein Assays can be used as tools to dissect and gain greater understanding of the mechanisms guiding reprogramming and to further characterize individual established iPSC lines.
authors.affiliation: Life Technologies Corporation, Carlsbad, California 92008, USA.
authors.forename: David
authors.initials: D
authors.lastname: Ruff
authors.forename: Chad
authors.initials: C
authors.lastname: MacArthur
authors.forename: Ha
authors.initials: H
authors.lastname: Tran
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Bergseid
authors.forename: Jack
authors.initials: J
authors.lastname: Tian
authors.forename: Mark
authors.initials: M
authors.lastname: Shannon
authors.forename: Shiaw-Min
authors.initials: SM
authors.lastname: Chen
authors.forename: Andrew
authors.initials: A
authors.lastname: Fontes
authors.forename: Louise
authors.initials: L
authors.lastname: Laurent
authors.forename: Elana
authors.initials: E
authors.lastname: Swartzman
authors.forename: Antje
authors.initials: A
authors.lastname: Taliana
authors.forename: Mahendra
authors.initials: M
authors.lastname: Rao
authors.forename: Pauline T
authors.initials: PT
authors.lastname: Lieu
mesh_terms: D054337:Cell Dedifferentiation; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D011506:Proteins