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Published on: January 22, 2013
Multiplex RT-PCR-Based Sequencing Assay to Detect Kidney Cancer-Specific Splice Variants in Tumor Tissues and Plasma
Kapil K Avasthi1, Kendrick H Yim2, Youngchul Kim3
1Department of Tumor Microenvironment & Metastasis, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA.
Molecular Diagnosis & Therapy
|July 6, 2026
Summary
This study introduces a new assay to detect specific splice variants (SVs) in clear cell renal cell carcinoma (ccRCC) patients. This method shows promise for non-invasive monitoring of ccRCC recurrence using blood samples.
Area of Science:
- Oncology
- Molecular Biology
- Genomics
Background:
- Clear cell renal cell carcinoma (ccRCC) lacks sensitive biomarkers for early detection of recurrence and monitoring.
- Recurrent splice variants (SVs) present a promising avenue for detecting tumor-specific signals in circulating blood.
Purpose of the Study:
- To develop and validate a multiplex assay for quantifying ccRCC-specific splice variants (SVs) in both tumor tissue and plasma.
- To assess the translational potential and clinical applicability of these SVs for non-invasive molecular profiling.
Main Methods:
- A 56-target multiplex reverse transcription-polymerase chain reaction (RT-PCR) panel was designed to amplify 27 ccRCC-associated SVs, 24 wild-type transcripts, and 5 housekeeping genes.
- SVs were validated using nanopore long-read sequencing, and analytical sensitivity was assessed with synthetic oligos.
- The assay's translational potential was evaluated in 47 matched tumor-plasma pairs, analyzing SV counts for detectability, concordance, stability, and robustness.
Main Results:
- Long-read sequencing confirmed cell line-specific isoform patterns. The assay demonstrated strong analytical performance with linear quantification and a low limit of detection.
- Several SVs, including TTCB-SV, MVK-beta-SV, MVK-alpha-SV, and MCCC-SV, showed high tumor-to-plasma concordance (0.87-0.98).
- Downsampling analyses confirmed robustness, with key SVs retaining high detectability even at reduced plasma sequencing depth.
Conclusions:
- The developed multiplex RT-PCR sequencing assay enables sensitive detection of ccRCC-associated SVs in both tumor RNA and plasma.
- The assay's high analytical fidelity, tumor-plasma concordance, and robustness support its advancement for non-invasive molecular profiling.
- This approach holds potential for longitudinal monitoring of ccRCC patients.
