Influence of different level of haemolysis on microRNAs for the early diagnosis of colorectal cancer

Introduction: microRNAs (miRNAs) are small non-coding RNAs involved in the development of various cancers. From our discovery study we identified a set of plasma circulating miRNAs associated with colorectal cancer lesions (CRC). However, significant variation in miRNA expression can be related to haemolysis (rupturing of erythrocytes) occurring during sampling and handling procedures [1,2]. Materials and methods: we set-up an in-vitro controlled haemolysis experiment to investigate if miRNAs expression is influenced by different level of haemolysis. Red blood cells (%RBCs) were used to artificially introduce haemolysis in a heamolysis-free plasma sample (S12) by performing 10 serial dilutions (range: 0.002% - 1% v/v) and single RT-qPCR was used for miRNAs quantification. We evaluated the relevance of the expression changes between sample S12 and the other samples (S11-S01) by computing, for each miRNA, the 95% Simultaneous Confidence Interval (SCI) [3] of the Relative Quantity (RQ) {RQ=2-ΔΔCt, where ΔΔCt=ΔCt(Sx) – ΔCt(S12) and ΔCt= Ct(miRNA) - Ct(ref) and Ct(ref) is the average of the miRNAs identified as reference by our procedure [4]}. By adopting the two-fold threshold rule, miRNAs were considered statistically influenced by haemolysis if the upper limit of the 95% SCI(RQ) was ≥ 2 or the lower limit of the 95% SCI(RQ) was ≤ 0.5. Results and conclusion: miRNAs known as haemolysis-related in literature were confirmed also in our experiment whereas our reference miRNAs seem to be not influenced by haemolysis. Similar results were obtained for the majority of our candidate miRNAs suggesting those as potential haemolysis-independent biomarker for early detection of CRC. Acknowledgements: This work was supported by grant from Associazione Italiana per la Ricerca sul Cancro (AIRC) (Grant No. 12162). References [1] Kirschner MB, et al. PlosOne. 2011; 6:e24145 [2] Appierto V, et al. Bioanalysis. 2014; 6:1215-26. [3] Pizzamiglio S, et al. Oncol Rep. 2010;23:853-60 [4] Verderio P, et al. Anal Biochem. 2014. 461C:7-9