Fondazione Italiana Fegato - ONLUS, Liver Research Center, AREA Science Park, Basovizza, Trieste, Italy.
Universitas Hasanuddin, Faculty of Medicine, Makassar, Indonesia.
Sci Rep. 2020 Feb 21;10(1):3128. doi: 10.1038/s41598-020-59682-0.
Normalization procedures for the qRT-PCR analysis of miRNA in biological samples are recommended to reduce the variability caused by pre-analytical factors. Since there is no universal standardized normalization strategy for miRNA qRT-PCR studies, we conducted a throughout study to evaluate a panel of small non-coding RNAs (sncRNAs) as reference gene candidate for biomarker studies in serum samples of patients with hepatocellular carcinoma (HCC). Five sncRNAs (miR-1280, miR-1275, SNORD-116, SNORD-68, and U6) were chosen as candidate of reference genes. This study included 122 patients with HCC and was organized into a "pilot phase" consisting of 20 serum samples of HCC patients, and a "validation phase" of 102 patients. Expression level of these candidates were analyzed by qRT-PCR. Assessment of gene stability was performed using four different integrative platforms (geNorm NormFinder, Bestkeeper, and the Delta Ct method). To determine the gene stability during the follow-up of the patient, we extend the analysis of the validation cohort at T1 (1 month after treatment) and T2 (6 month after treatment). MiR-1280 was identified as the most stably expressed reference gene in both pilot and validation phase also during the follow-up. MiR-1280 appears a reliable reference gene candidate in biomarker studies.
建议对 miRNA 的 qRT-PCR 分析进行标准化处理，以减少由预分析因素引起的变异性。由于 miRNA qRT-PCR 研究没有通用的标准化标准化策略，我们进行了一项全面的研究，以评估一组小非编码 RNA（sncRNA）作为候选参考基因，用于肝癌（HCC）患者血清样本中的生物标志物研究。选择了 5 个 sncRNA（miR-1280、miR-1275、SNORD-116、SNORD-68 和 U6）作为候选参考基因。这项研究包括 122 名 HCC 患者，并分为“试点阶段”（由 20 名 HCC 患者的血清样本组成）和“验证阶段”（102 名患者）。通过 qRT-PCR 分析这些候选者的表达水平。使用四种不同的综合平台（geNorm NormFinder、Bestkeeper 和 Delta Ct 方法）评估基因稳定性。为了确定患者随访期间的基因稳定性，我们在 T1（治疗后 1 个月）和 T2（治疗后 6 个月）时扩展了验证队列的分析。miR-1280 在试点和验证阶段以及随访期间均被确定为最稳定的参考基因。miR-1280 似乎是生物标志物研究中可靠的参考基因候选物。
