Applying Subtractive Hybridization Technique to Enrich and Amplify Tumor-Specific Transcripts of Esophageal Squamous Cell Carcinoma

Abstract

Subtractive hybridization (SH) as an efficient and powerful approach can be applied to isolate differentially expressed transcripts as well as detect of involved mRNAs in various cellular processes, particularly diseases and malignancies. This procedure leads to the enrichment of specific low copy transcripts of tumor cells. Having developed a new approach for SH to isolate tumor specific transcripts, we facilitated discovery of uniquely expressed genes in esophageal squamous cell carcinoma (ESCC). Total RNA was extracted from the fresh tumoral and their adjacent normal tissues, and purified using the Switch Mechanism At the 5' end of Reverse Transcript (SMART) method. Following cDNA synthesis of normal mRNAs using magnetic beads, it was hybridized with tumor mRNAs. To enhance efficiency of subtraction, hybridization was repeated three rounds. Finally, amplification of subtracted tumor-specific transcripts was carried out using in vitro transcription. The subtracted tumoral mRNAs was analyzed quantitatively using real-time PCR for both tumor-specific and housekeeping genes. The subtracted mRNA was confirmed as tumor-specific mRNA pool using RT-PCR and quantitative real-time PCR assessment. The elevated level of tumor-specific transcripts such as MAGE-A4 and CD44 as well as declined copy number of housekeeping genes such as GAPDH, β actin and β2-microglobulin, were confirmed in subtracted tumoral mRNA. The presence of tumor genes was confirmed after the SH procedure. The designed SH method in combination with SMART technique can isolate and amplify high quality tumor-specific transcripts even from small amount of tumor tissues. Removal of common transcripts from the extracted tumoral mRNAs using SH, leads to the enrichment of tumor-specific transcripts. The isolated transcripts are of interest because of their probable roles in ESCC progression and development. In addition, these tumor-specific mRNAs can be applied for future vaccine cancer studies.