A simple and efficient method for the isolation of differentially expressed genes.
A simple and reproducible general approach for the isolation of differentially expressed genes is described. Digestion of cDNAs with a class IIs restriction endonuclease produces fragments with every combination of possible bases in the cohesive ends. Under stringent conditions, the specific ligation of adaptors with perfectly complementary overhangs partitions the cDNA fragments into non-overlapping subpopulations. Internal cDNA restriction fragments are exponentially amplified by adaptor primer PCR and visualised by non-denaturing polyacrylamide gel electrophoresis. The power of the technology was demonstrated using a rat model of pressure-induced left-ventricular hypertrophy (LVH). A set of 29 fragments, derived from a sample (6 %) of the possible adaptor pool combinations, displayed apparent differential expression. The differential expression of 19 (66 %) were confirmed by Northern blot analysis. Sequence analysis identified both genes known to be upregulated in LVH, and novel genes. The fidelity of adaptor ligation was demonstrated by the isolation of known gene fragments by appropriate adaptor combinations. The spiking of mRNA populations with known amounts of a synthetic mRNA demonstrated a current sensitivity equivalent to the detection of transcripts expressed at the level of as little as 1 in 10,000 molecules.
Mahadeva
H
H
Department of Cardiology, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Groby Road, Leicester, LE3 9QP, UK.
Starkey
M P
MP
Sheikh
F N
FN
Mundy
C R
CR
Samani
N J
NJ
D000818:Animals; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005821:Genetic Techniques; D017379:Hypertrophy, Left Ventricular / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific