Evaluation and optimization of different cf‐RNA extraction methods for Alzheimer‐related genes detection

Abstract

AbstractBackgroundCell‐free RNA (cf‐RNA) in plasma has become a fascinating less‐invasive diagnostic approach for Alzheimer’s Disease (AD) since brain‐originated mRNA and microRNA can enter the bloodstream through the blood‐brain barrier. Studying cf‐RNA expression in blood samples collected from AD‐patients and at‐risks patients allows further understanding of disease pathogenesis, contributes to the development of early diagnosis and prognosis methods. However, contaminants risks, low yield, and integrity can alter final cf‐RNA quality and concentration, challenging their application. This study aims to evaluate and optimize different cf‐RNA extraction protocols, as well as to detect the AD‐related transcripts in the final sample.MethodWe assessed different cf‐RNA extraction methods and evaluated hemolysis ‐ the main contamination threat to the final product. Blood samples were collected from healthy volunteers into designated collection tubes to compare their effects on hemolysis and RNA products. Plasma cf‐RNA was extracted using the Trizol and Zymo cf‐RNA kits, with two modifications to the Trizol protocol: addition of glycogen and with the use of Rneasy mini kit to increase cf‐RNA yield. Among 30 blood samples were collected, 12 were treated with Zymo, six with Trizol, eight with Trizol w/glycogen and four with Rneasy. cf‐RNA was then converted to cDNA and examined with PCR and qPCR.ResultNorgen tubes showed a lower hemolysis level compared to Streck tubes. The yield of cf‐RNA from the Zymo kit was about 5‐18 µg/ml. The Trizol protocol resulted in almost no detectable cf‐RNA. The addition of glycogen significantly increased the yield to a comparable level with the Zymo kit (4‐15 µg/ml). No genomic DNA contamination was detected. Despite high cf‐RNA yield from the modified Trizol protocol, the housekeeping gene Beta‐actin (ACTB) or other AD‐related transcripts (CNTF and MAG) could not be detected. Using cDNA obtained from the Zymo kit, ACTB was reliably detected, but AD‐related transcripts’ results varied. cDNA concentration was high‐correlated with the melt curve analysis of ACTB qPCR, suggesting Zymo kit obtained cDNA had high integrity.ConclusionOverall, the Zymo kit is superior to the Trizol‐based protocols in both cf‐RNA yield and integrity. The final sample showed detectable levels of ACTB and AD‐related transcripts.