Effect of different normalization approaches on the levels of serum miRNAs.

<p>(<b>A</b>) Serum samples from healthy controls, ulcerative colitis patients and colon cancer patients were divided into three groups (n =5 in each group). After the initial denaturation steps, 1, 2 and 4 attomole of synthetic artificial miRNA (5’-GUGGAUUCCGUCUCGUUAG-3’) were spiked into 100 μL of serum of each group (1 attomole for control group, 2 attomole for ulcerative colitis group and 4 attomole for colon cancer group). After isolation of total RNA, the levels of artificial miRNA were assessed by RT-qPCR assay and were normalized to serum volume, let-7d/g/i, U6 or miR-191, respectively. Relative levels were calculated using the 2^-△△Cq method and were shown by dot plots. Significance was calculated by t-test. (<b>B</b>) Expression levels of miR-25, miR-214, miR-223 and miR-483-5p were measured in serum from cancer patients (n = 84) and healthy controls (n = 41) by RT-qPCR and were normalized to serum volume, let-7d/g/i, U6 or miR-191. Relative levels were calculated using the 2^-△△Cq method and are presented as mean fold changes ± standard errors. Significance was calculated by t-test.