Relative quantity and variation associated with each candidate reference gene. (a) Quantity of candidate reference miRNAs in colorectal tumour (n = 35) and normal (n = 39) tissues as expressed as quantification cycle (Cq) values. Boxplots depict median lines, interquartile-range boxes and outliers (*). Error bars represent range of values. No significant difference (p > 0.05, t-test) was found within all reference genes between tumour and normal tissues, thus supporting further evaluation of these genes as references. (b) Variation associated with each candidate reference gene. There was a significant difference in variance (p < 0.001, Bartlett's test) associated with each reference gene indicating differing stabilities. RNU48 and Z30 showed greater variance than miR-16, miR-345 and miR-425.