Improved method for detecting differentially expressed genes using cDNA indexing.
In cDNA indexing, differentially expressed genes are identified by the display of specific, corresponding subsets of cDNA. Subdivision of the cDNA population is achieved by the sequence-specific ligation of adapters to the overhangs created by class IIS restriction enzymes. However, inadequate specificity of ligation leads to redundancy between different adapter subsets. We evaluate the incidence of mismatches between adapters and class IIS restriction fragments during ligation and describe a modified set of conditions that improves ligation specificity. The improved protocol reduces redundancy between amplified cDNA subsets, which leads to a lower number of bands per lane of the differential display gel, and therefore simplifies analysis. We confirm the validity of this revised protocol by identifying five differentially expressed genes in mouse duodenum and ileum.
Shaw-Smith
C J
CJ
Gastroenterology Section, Imperial College School of Medicine, Hammersmith Hospital, London, England, UK.
Coffey
A J
AJ
Huckle
E
E
Durham
J
J
Campbell
E A
EA
Freeman
T C
TC
Walters
J R
JR
Bentley
D R
DR
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics*; D004386:Duodenum; D015870:Gene Expression; D007082:Ileum; D051379:Mice; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D009841:Oligonucleotides; D012333:RNA, Messenger; D011088:DNA Ligases