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Updated: Aug 18, 2026
The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Published on: January 22, 2013
Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma
1Department of Urology, Institute of Urology, First Affiliated Hospital of Beijing Medical University, Beijing 100034, China. zhanqian@public3.bta.net.cn
Objective:
To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization.
Methods:
Polyadenylated RNA [Poly (A)+ RNA] was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors I and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes.
Results:
A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400 bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18.
Conclusions:
The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.
Insights
Researchers developed a renal cell carcinoma (RCC) cDNA subtractive library to identify novel genes. This library successfully identified new genes, including RCC18, which is highly expressed in RCC tumors, aiding in understanding cancer development.
Area of Science:
- Molecular biology
- Oncology
- Genomics
Background:
- Renal cell carcinoma (RCC) is a significant health concern.
- Identifying novel genes specific to RCC is crucial for understanding its pathogenesis.
- Suppression subtractive hybridization (SSH) is a powerful technique for identifying differentially expressed genes.
Observation:
- A subtractive cDNA library was constructed using SSH from RCC and normal kidney tissues.
- The library yielded 350 positive clones, with 85 containing inserts after random analysis.
- Sequence analysis revealed six unique, novel genes, including RCC18, which was highly expressed in RCC.
Findings:
- A highly efficient human RCC cDNA subtractive library was successfully established.
- Novel gene RCC18 was identified and confirmed to be highly expressed in RCC tissues via Northern blot.
- The full-length sequence of the novel gene RCC18 was obtained using SMART RACE.
Implications:
- The constructed library provides a foundation for large-scale screening of RCC-specific genes.
- The identified novel genes, like RCC18, offer insights into RCC occurrence and development mechanisms.
- This research facilitates the discovery of potential oncogenes and tumor suppressor genes for RCC.
