Improper preanalytical processes on peripheral blood compromise RNA quality and skew the transcriptional readouts of mRNA and LncRNA

Abstract

Genetic and epigenetic reprogramming caused by disease states in other tissues is always systemically reflected in peripheral blood leukocytes (PBLs). Accurate transcriptional readouts of Messenger RNA (mRNA) and Long non-coding RNA (lncRNA) in peripheral blood leukocytes are fundamental for disease-related study, diagnosis and treatment. However, little is known about the impact of preanalytical variables on RNA quality and downstream messenger RNA and Long non-coding RNA readouts. In this study, we explored the impact of RNA extraction kits and timing of blood placement on peripheral blood leukocyte-derived RNA quality. A novel enhanced evaluation system including RNA yields, purity, RNA integrity number (RIN) values and β-actin copies was employed to more sensitively identify RNA quality differences. The expression levels of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease (COPD) or triple-negative breast cancer (TNBC) were measured by Quantitative reverse transcription polymerase chain reaction (qRT–PCR) to investigate the impact of RNA quality on transcriptional readouts. Our results showed that the quality of RNA extracted by different kits varies greatly, and commercial kits should be evaluated and managed before batch RNA extraction. In addition, the quality of extracted RNA was highly correlated with the timing of blood placement, and the copy number of β-actin was significantly decreased after leaving blood at RT over 12 h. More importantly, compromised RNA leads to skewed transcriptional readouts of informative mRNAs and Long non-coding RNAs in patients with chronic obstructive pulmonary disease or triple-negative breast cancer. These findings have significant implications for peripheral blood leukocyte-derived RNA quality management and suggest that quality control is necessary prior to the analysis of patient messenger RNA and Long non-coding RNA expression.