Expression analysis of subtractively enriched libraries (EASEL): a widely applicable approach to the identification of differentially expressed genes.
A variety of methods for high throughput analysis of differential gene expression has been developed over the past years. We have implemented the EASEL technique that adds flexibility, efficiency and wide-applicability to these methods. The EASEL procedure is unique as it integrates several well established techniques and thereby offers a combination of subtractive hybridization of 3' cDNA ends with macroarrays analysis and Serial Analysis of Gene Expression (SAGE). In addition, once a set of interesting, differentially expressed genes is identified, the material required for follow up studies to test the hypothesis that the gene is truly involved in the process of interest is readily available. In this report, we first present a step-by-step validation of the procedure, since several of the incorporated steps had to be tailored to meet specific requirements and implied drastic modifications of the original methods. Secondly, we applied EASEL to the identification of up-regulated gene products in the outflow tract region of the embryonic rat heart. Here we provide evidence that at least two among the differentially expressed genes detected, follistatin-like protein gene and membrane type 1-metallo proteinase gene, are selectively up-regulated in the outflow tract, suggesting their involvement in the development of this region during embryogenesis.
Lombardi
M Paola
MP
Experimental and Molecular Cardiology Group, Cardiovascular Research Institute Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands.
van den Hoff
Maurice J B
MJ
Ruijter
Jan M
JM
Luijerink
Marjanka
M
Buffing
Anita A
AA
Markman
Marry W
MW
Moorman
Antoon F M
AF
Lekanne Deprez
Ronald H
RH
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis; D038702:Follistatin-Related Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006321:Heart / Q000196:embryology / Q000502:physiology; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D051381:Rats; D015203:Reproducibility of Results
D018076:DNA, Complementary; D038702:Follistatin-Related Proteins; C448719:Fstl1 protein, rat