Expanding applications of protein analysis using proximity ligation and qPCR.
The correlation of gene and protein expression changes in biological systems has been hampered by the need for separate sample handling and analysis platforms for nucleic acids and proteins. In contrast to the simple, rapid, and flexible workflow of quantitative PCR (qPCR) methods, which enable characterization of several classes of nucleic acid biomarkers (i.e. DNA, mRNA, and microRNAs), protein analysis methods such as Western blotting are cumbersome, laborious, and much less quantitative. However, TaqMan(R) Protein Assays, which use the proximity ligation assay (PLA) technology, now expand the range of qPCR applications to include the direct detection of proteins through the amplification of a surrogate DNA template after antibody binding. Here we describe an integrated qPCR approach for measuring relative changes in gene and protein expression from the same starting sample and on a single analytical platform that pairs TaqMan Gene Expression (GEx) Assays with TaqMan Protein Assays. We have monitored the changes in mRNA, microRNA, and protein expression of relevant biomarkers in the pluripotent human embryonal carcinoma cell line, NTERA2, upon differentiation to neuronal cells. In addition, TaqMan Protein Assays have been used to monitor protein expression in induced pluripotent stem cells (iPSC) that have been reprogrammed from human somatic cells. The data presented establishes a general paradigm utilizing real-time PCR instruments and reagents for studying the relationship between the stem cell transcriptome and proteome.
Swartzman
Elana
E
Applied Biosystems, Part of Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 94404, USA. Elana.Swartzman@lifetech.com
Shannon
Mark
M
Lieu
Pauline
P
Chen
Shiaw-Min
SM
Mooney
Chad
C
Wei
Eric
E
Kuykendall
Julie
J
Tan
Rouying
R
Settineri
Tina
T
Egry
Levente
L
Ruff
David
D
D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D019411:Clinical Laboratory Techniques; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014212:Tretinoin / Q000494:pharmacology
D035683:MicroRNAs; D012333:RNA, Messenger; D014212:Tretinoin