Housekeeping genes for studies of plasma microRNA: A need for more precise standardization

Plasma microRNAs (miRNAs) are promising biomarkers for many forms of cancer in humans; however, a fundamental concern is the lack of standardization in current data acquisition and reporting. Part of this problem lies in the use of numerous, different housekeeping genes (HKG) for the acquisition of real-time polymerase chain reaction data. This existing practice of using different HKGs generally is accepted, but reproducibility of data for comparison and validation between different laboratories calls for improvement. The need for data reproducibility standardization is crucial. An ideal plasma HKG (1) should be expressed in all samples, (2) have medium-to-high levels of expression, and (3) have consistently measurable levels of expression.

Total RNA was extracted from 200-μL plasma samples via a modified miRNeasy (QIAGEN) extraction technique with yeast carrier. Total RNA purity was assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific). The cycle threshold (Ct) was fixed at 0.03 for all samples. We investigated 10 potential HKGs based both on reports in the literature and our previous data. The potential HKGs were Let-7a, Let-7d, Let-7g, miR-16, RNU6, RNU48, miR-191, miR-223, miR-484, and miR-520d-5p. Once all samples were run for each potential HKG, the mean Ct and SD was calculated for all sample groups, allowing for comparison among HKGs.

We screened 380 miRNAs by using microfluidic array technology (Applied Biosystems) in a discovery cohort of 20 colorectal cancer (CRC) patients, 10 patients each with breast cancer (BC), lung cancer (LC), pancreatic cancer (PC), 11 patients with colorectal adenoma, and 12 controls. The mean Ct and SD was calculated for RNU6, miR-520d-5p, miR-16, miR-191, miR-223, and miR-484, which were expressed in all samples. Let-7a, Let-7d, Let-7g, and RNU48 were only expressed in 26%, 7%, 10%, and 8% of samples, respectively, and therefore were deemed to be insufficiently reliable HKGs. Only miRNAs with >50% expression were included in this statistical analysis. U6 and miR-520d-5p had the most consistent Ct as well as the least SD. The use of both RNU6 and 520d-5p as HKGs provided reliable results.

Among HKGs that were expressed in all samples, we suggest that RNU6 and miR-520d-5p were the best candidates for HKGs for studies of plasma miRNA because of the consistent and high Ct in all samples and a very narrow, reproducible SD.