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.

Dry blood spot (DBS) technology has been widely used since the 1960's for the detection of protein biomarkers associated with various disease states. In this manuscript we report a revised approach using DBS samples to extract total RNA for use in downstream multiplex RNA detection methodology (Nanostring). To accomplish this objective, we have used commercially available supplies, kits, and equipment to ensure that the procedure described in this report can be adopted by any laboratory. The methods described in this report allow for the extraction of high‐quality, total RNA from a minimal volume (∼200 µl) of DBS spots. The isolated RNA can be analyzed using a multiplex, Nanostring system to yield results for up to 800 RNA targets. Additional bioinformatics and pathway annotation can be conducted to determine changes in biological signaling pathways. © 2023 Wiley Periodicals LLC. Basic Protocol: RNA extraction from DBS for multiplex RNA nanostring analysis Support Protocol 1: RNA extraction from PAXgene blood Support Protocol 2: Concentration of DBS RNA