Magnetic Isolation, RNA Extraction, and Nanostring Analysis of Human Peripheral Blood Leukocytes Subsets.

Abstract

Advancements in multiplexed molecular biology techniques have allowed for blood samples and specific circulating blood leukocytes to be a useful sample source when examining systemic changes associated with changes in body weight, muscle injury, disease onset/progression, and other common conditions. One gap in the current scientific knowledge relates to the impact of changes in individual leukocyte subsets on the overall systemic response. While many studies have published data related to changes observed in a mixed population of circulating leukocytes (i.e., whole blood sample), few studies have identified which cell(s) are responsible for the overall change. Since it is established that leukocyte subsets respond differently to various experimental challenges, it may be possible to gain new insight regarding overall biological state. This has application to a variety of health, nutrition, and exercise intervention models. Despite the need to examine changes in mRNA expression levels in individual leukocyte subsets they are not always easy to isolate and perform mRNA analysis on. In this report we describe a method to magnetically isolate, stabilize RNA, and analyze 800+ mRNA in a single sample analysis. Further we compared total leukocyte and leukocyte subset (i.e., granulocytes, monocytes, and T-cells) mRNA expression to better understand how subset changes contribute to overall response. Examination of subset responses may provide targets for future intervention studies. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Automated magnetic isolation of granulocytes, monocytes, and T-cells Basic Protocol 2: RNA extraction from magnetically sorted granulocytes, monocytes, and T-cells Basic Protocol 3: Nanostring analysis of RNA extracted from magnetically sorted granulocytes, monocytes, and T-cells.