Identification and Cloning of RA-Regulated Genes by mRNA-Differential Display
互联网
217
An important key to understanding the function of retinoids is the determination of genes whose expression they regulate. In the past, several techniques including differential screening (
1
), subtractive hybridization (
2
), reverse transcrrptase-polymerase chain reaction (RT-PCR) analysis (
3
), and RNase protection (
4
) have been utilized to study specific differences in gene expression following RA treatment. Although these techniques have proven useful, they are not without limitations; differential screening and subtractive hybridization are both technically demanding and lengthy procedures relymg heavily on the quality of cDNA and genomic libraries used, RT-PCR and RNase protectton analyses provide only quantitative mformatton on previously characterized genes. Another drawback in the use of subtractive hybridization or differential screening is that these techniques only allow analyses of unidirectional, either upregulatton or downregulation, changes in gene expression in one of the samples being compared. The recent development of a novel PCRbased procedure, mRNA-differential display (DD), provides a powerful alternative method that overcomes some of the shortfalls of other techniques (
5
,
6
). DD is unique in that it permits the simultaneous comparisons of both positive and negative alterations in gene expression between multiple samples (
see
Fig. 1 ).
Fig. 1.
Schematic representation of the steps involved in the isolation of retinoid-regulated genes using the differential display technique. The cloned producis isolated in step 6 can then be used for sequencing, Northern blotting, or screening of cDNA libraries. Pl, P2, and P3 correspond to fragments from RA induced mRNAs. P4 represents a PCR product from an mRNA that is downregulated.
