Impact of Washing Processes on RNA Quantity and Quality in Patient-Derived Colorectal Cancer Tissues

Abstract

Background: Colorectal cancer (CRC) is a common and lethal cancer worldwide. Extraction of high-quality RNA from CRC samples plays a key role in scientific research and translational medicine. Specimen collection and washing methods that do not compromise RNA quality or quantity are needed to ensure high quality specimens for gene expression analysis and other RNA-based downstream applications. We investigated the effect of tissue specimen collection and different preparation processes on the quality and quantity of RNA extracted from surgical CRC tissues. Materials and Methods: After surgical resection, tissues were harvested and prepared with various washing processes in a room adjacent to the operating room. One hundred fourteen tissues from 36 CRC patients were separately washed in either cold phosphate-buffered saline reagent (n = 34) or Dulbecco's modified Eagle's medium (DMEM; n = 34) for 2-3 minutes until the stool was removed, and unwashed specimens served as controls (n = 34). Six tissue specimens were washed and immersed in DMEM for up to 1 hour at 4°C. Before RNA extraction, all specimens were kept in the stabilizing reagent for 3 months at -80°C. RNA was extracted, and the concentration per milligram of tissue was measured. RNA quality was assessed using the RNA integrity number (RIN) value. Results: Different washing processes did not result in significant differences in RNA quantity or RIN values. In the six tissues that were washed and immersed in DMEM for 1 hour, RIN values significantly decreased. The quality of the extracted RNA from most specimens was excellent with the average RIN greater than 7. Conclusions: RNA is stable in specimens washed in different processes for short periods, but RIN values may decrease with prolonged wash times.