Premium
Identification and Verification of Changes in Gene and Protein Expression from Limited Samples
Author(s) -
Lin Emily,
Oh Kenneth,
Okino Steven,
Liu Ning,
Wang Yan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.880.5
Subject(s) - homeobox protein nanog , messenger rna , gene expression , gene , biology , lysis , microbiology and biotechnology , western blot , retinoic acid , computational biology , embryonic stem cell , genetics , induced pluripotent stem cell
Identification and validation of regulatory changes is challenging when analyzing small samples, especially when both gene and protein expression analysis is necessary. We developed a novel workflow that allows for screening up to 96 genes for differential mRNA expression, and validation that the protein expression level of selected targets are indeed changed. To validate our workflow we assessed changes that occur in NTera2 cells, a human stem cell model system, after four days of differentiation initiated by retinoic acid. NTera2 were grown in a 96‐well plate under control or differentiated conditions. One well each of control and differentiated cells were harvested to generate a cell lysate. A small portion of each lysate was analyzed for mRNA expression; we incorporated a pre‐amplification step to allow the analysis of a large panel of genes associated with pluripotency and differentiation. We observe differential expression of several genes, including NANOG and OCT4 that are down‐regulated in the differentiated cells. We then performed Western blot analysis, using the remaining cell lysate, to assess NANOG and OCT4 protein levels. We find that the level of protein for these targets is significantly decreased in the differentiated cells, consistent with the mRNA results. These findings demonstrate that (1) mRNA and protein analyses can be conducted from the same cell sample, and (2) a large panel of gene targets can be screened to identify candidates for subsequent verification by protein analysis. We envision that this workflow can enable streamlined analysis and verification of regulatory changes at both the mRNA and protein level in samples that are typically refractory to such analysis.