Differential subtraction display: a unified approach for isolation of cDNAs from differentially expressed genes.
We have developed a novel efficient approach, termed differential subtraction display, for the identification of differentially expressed genes. Several critical parameters for the reproducibility and enhanced sensitivity of display, as well as steps to reduce the number of false positive cDNA species, have been defined. These include- (a) use of standardized oligo(dT)-primed cDNA pools rather than total RNA as the starting material for differential display, (b) critical role of optimal cDNA input for each distinct class of primers, (c) phenomena of primer dominance and interference, and (d) design of a novel set of enhanced specificity anchor primers. Introduction of an efficient subtractive hybridization step prior to cloning of cDNA species enriches the bona fide cDNA species that are either exclusively present in one sample (+/-) or show altered expression (up-/down-regulation) in RNA samples from two different tissues or cell types. This approach, in comparison to differential display, has several advantages in terms of reproducibility and enhanced sensitivity of display coupled to the cloning of enriched bona fide cDNA species corresponding to differentially expressed RNAs.
Pardinas
J R
JR
Skin Biology Research Center, Johnson & Johnson CPWW, Skillman, New Jersey 08558-9418, USA.
Combates
N J
NJ
Prouty
S M
SM
Stenn
K S
KS
Parimoo
S
S
D000818:Animals; D019921:Biotinylation; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*; D015870:Gene Expression; D005786:Gene Expression Regulation; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA