Enhancing negative control selection: A comparative analysis of random and targeted sampling techniques for obtaining High-Quality RNA from normal breast tissue.

Abstract

Molecular profiling is a crucial aspect of cancer therapy selection, underscoring the necessity for representative sampling of both tumor and normal tissues. While much attention has been given to representative tumor sampling, there has been a notable lack of exploration into the issue of poor RNA quality in normal breast tissue processing. Normal breast tissue from the same patient is often used as a negative control for most "-omics" experiments. RNA extracted from normal breast tissues frequently contains nucleic acids from surrounding adipocytes, endothelial cells, and immune cells, leading to a low representation of ductal elements and skewed results. Therefore, ensuring a complete representation of breast glandular tissue is imperative. The study aimed to investigate the variations in RNA enrichment between a random sampling technique and a targeted sampling approach when visually selecting normal breast tissue sections as negative controls for "-omics" experiments. Fifteen female breast cancer subjects who underwent Modified Radical Mastectomy were selected for the study. Normal Breast tissue was visually examined, and samples were collected from random fat pockets (random sampling) and fibromuscular grey-white streak areas (targeted sampling). RNA was isolated, followed by spectrophotometric analysis, agarose gel electrophoresis and Agilent Tape station analysis. Histopathological assessments and a gene expression study for housekeeping genes were performed on both subsets. Tissues collected through targeted sampling exhibited significantly higher RNA quality than those obtained via random sampling. Histopathological analysis revealed cellular areas abundant in terminal ductular units within the targeted samples, and a final validation qPCR showed that the targeted samples were the most representative of normal breast glandular tissue. The comparative analysis of the two sampling methods clearly indicates that the targeted approach, with its superior accuracy and reliability, is the more practical choice for obtaining representative normal breast glandular tissue for "-omics" experiments.