Identification of reference genes for circulating microRNA analysis in colorectal cancer
Quantitative real-time PCR (qPCR) is the most frequently used method for measuring expression levels of microRNAs (miRNAs), which is based on normalization to endogenous references. Although circulating miRNAs have been regarded as potential non-invasive biomarker of disease, no study has been performed so far on reference miRNAs for normalization in colorectal cancer. In this study we tried to identify optimal reference miRNAs for qPCR analysis across colorectal cancer patients and healthy individuals. 485 blood-derived miRNAs were profiled in serum sample pools of both colorectal cancer and healthy control. Seven candidate miRNAs chosen from profiling results as well as three previous reported reference miRNAs were validated using qPCR in 30 colorectal cancer patients and 30 healthy individuals, and thereafter analyzed by statistical algorithms BestKeeper, geNorm and NormFinder. Taken together, hsa-miR-93-5p, hsa-miR-25-3p and hsa-miR-106b-5p were recommended as a set of suitable reference genes. More interestingly, the three miRNAs validated from 485 miRNAs are derived from a single primary transcript, indicting the cluster may be highly conserved in colorectal cancer. However, all three miRNAs differed significantly between healthy individuals and non-small cell lung cancer or breast cancer patients and could not be used as reference genes in the two types of cancer.
[…] Statistical analysis was carried out using the GraphPad Prism 5 using two-tailed Student’s test. Data were present as means ± SE (standard error). P value of less than 0.05 was considered statistically significant. Mean values were obtained from SPSS software, version19.0 (SPSS Inc., Chicago, IL, USA). The stability of the candidate reference genes was evaluated by the computer program BestKeeper, geNorm and NormFinder. In the BestKeeper software, data observations are in the form of raw threshold cycles, while fold change were used as input data in both geNorm and NormFinder. […]
Breast Neoplasms, Carcinoma, Non-Small-Cell Lung, Neoplasms, Colorectal Neoplasms
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