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Updated: Jul 7, 2026
The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Published on: January 22, 2013
[Screening of differentially expressed genes in human renal cell carcinoma using suppression subtractive
1Center for Laboratory Medicine, First Affiliated Hospital of School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China. ywang00@sohu.com
Objective:
To suppress COL1A1 and COL3A1 gene expressions in human skin fibroblasts (HSFs) by means of RNA interference (RNAi).
Methods:
SSH was performed in two directions to isolate the differentially expressed genes between human a RCC cell line RLC-310 and a normal renal cell line HK-2 (ATCC). The cDNAs obtained from the final nested PCR were directly inserted into T/A cloning vector to establish a subtractive cDNA library of specifically or highly expressed genes in RCC. Reverse Northern dot blotting was performed to screen the truly differentially expressed genes, and 200 positive genes were randomly selected for sequencing.
Results:
The two-directional subtractive libraries contained more than 1200 clones, and 213 positive clones were obtained using reverse Northern blotting. Sequence analysis of these clones identified 144 differentially expressed genes, including 67 up-regulated and 77 down-regulated genes, in which 14 novel ESTs and 21 functionally unknown genes were found. Cluster analysis indicated the involvement of the sequenced genes in cell growth, cell adhesion and apoptosis.
Conclusion:
Reliable subtractive cDNA libraries of human RCC have been constructed successfully with SSH. The identification of the gene expression profile in RCC may help clarify the mechanism of tumorigenesis and development of RCC, and also sheds light on new targets for prevention, diagnosis and therapy of this malignancy.
Insights
This study successfully constructed subtractive cDNA libraries for renal cell carcinoma (RCC). The identified gene expression profile offers insights into RCC mechanisms and potential therapeutic targets.
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Context:
- Renal cell carcinoma (RCC) is a significant malignancy with complex genetic underpinnings.
- Understanding gene expression profiles is crucial for elucidating tumorigenesis.
- Subtractive hybridization (SSH) is a powerful technique for identifying differentially expressed genes.
Purpose:
- To construct subtractive cDNA libraries for human RCC.
- To identify genes differentially expressed in RCC compared to normal renal cells.
- To analyze the functional roles of identified genes in RCC.
Summary:
- Two-directional SSH yielded over 1200 clones, with 213 positive hits identified via reverse Northern blotting.
- Sequence analysis revealed 144 differentially expressed genes in RCC, including novel expressed sequence tags (ESTs) and functionally unknown genes.
- Cluster analysis indicated these genes are involved in critical cellular processes such as growth, adhesion, and apoptosis.
Impact:
- Successful construction of reliable SSH-based cDNA libraries for RCC.
- The identified gene expression profile aids in understanding RCC mechanisms.
- Provides potential new targets for RCC prevention, diagnosis, and therapy.
