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Analysis of differential gene expression in the kidney by differential cDNA screening, subtractive cloning, and mRNA
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421, USA.
Abstract:
It is becoming increasingly evident that significant changes in gene expression occur during the course of disease progression in both genetic and nongenetic kidney diseases. Knowledge of the differentially expressed genes may yield information about the abnormal biochemical events that occur in the initiation and pathogenesis of these diseases. The purpose of this review is to provide an overview of some of the current approaches for identifying and analyzing differentially expressed genes. The power of these techniques lies in their their ability to detect differences in the levels of specific mRNAs in the diseased compared to the nondiseased kidney without prior knowledge of their identity. The three basic techniques considered are differential cDNA library screening, subtracted cDNA libraries, and PCR-based differential display. Emphasis is placed on cDNA library construction and differential screening. Also reviewed are the analysis of differentially expressed cDNAs by Southern and Northern blot hybridization, S1-protection, RT-PCR, DNA sequencing, and DNA sequence analysis.
Insights
Understanding gene expression changes in kidney disease is crucial for identifying disease mechanisms. This review covers methods for detecting differentially expressed genes, aiding in the study of kidney disease pathogenesis.
Area of Science:
- Nephrology
- Molecular Biology
- Genomics
Background:
- Significant alterations in gene expression are observed during the progression of both genetic and non-genetic kidney diseases.
- Identifying these differentially expressed genes offers insights into the abnormal biochemical events underlying disease initiation and pathogenesis.
Purpose of the Study:
- To provide an overview of current methodologies for identifying and analyzing differentially expressed genes in kidney diseases.
- To highlight techniques capable of detecting differences in messenger RNA (mRNA) levels between diseased and non-diseased kidney tissues without prior gene identification.
Main Methods:
- Differential complementary DNA (cDNA) library screening
- Subtracted cDNA libraries
- Polymerase Chain Reaction (PCR)-based differential display
- Emphasis on cDNA library construction and differential screening
- Analysis of differentially expressed cDNAs via Southern and Northern blot hybridization, S1-protection, reverse transcription PCR (RT-PCR), DNA sequencing, and DNA sequence analysis.
Main Results:
- The reviewed techniques enable the detection of specific mRNA level differences in diseased versus non-diseased kidneys.
- These methods facilitate the discovery of novel genes involved in kidney disease pathogenesis.
Conclusions:
- Current molecular techniques offer powerful tools for dissecting the molecular underpinnings of kidney diseases.
- Further application of these methods can advance our understanding of kidney disease progression and potentially lead to new therapeutic targets.