Combination of PCR subtraction and cDNA microarray for differential gene expression profiling.
PCR subtraction hybridization has been used effectively to enrich and single out differentially expressed genes. However identification of these genes by means of cloning and sequencing individual cDNAs is a tedious and lengthy process. In this report, an attempt has been made to combine the use of PCR select cDNA subtraction hybridization and cDNA microarrays to identify differentially expressed genes using a nonradioactive chemiluminescent detection method. mRNA from human prolactin (hPRL) or human prolactin antagonist (hPRL-G129R) treated and non-treated breast cancer cells was isolated, and cDNAs were synthesized and used for the PCR subtraction to enrich the differentially expressed genes in the treated cells. The PCR-amplified and subtracted cDNA pools were purified and labeled using the digoxigenin method. Labeled cDNAs were hybridized to a human apoptosis cDNA microarray membrane and identified by chemiluminescence. The results suggest that the strategy of combining all three methods will allow for a more efficient, nonradioactive way of identifying differentially expressed genes in target cells.
Beck
M T
MT
Clemson University, SC, USA.
Holle
L
L
Chen
W Y
WY
D001709:Biotechnology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases