Data Normalization of Urine miRNA Profiling from Head and Neck Cancer Patients Treated with Cisplatin

The microRNA (miRNA) expression profile by qRT-PCR depends directly on the most appropriate normalization strategy adopted; however, currently there is no universally adequate reference gene. Therefore, this study aimed to determine, considering RNA-Seq results, the most adequate endogenous normaliz...

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Bibliographic Details
Published in:International journal of molecular sciences 2023-06, Vol.24 (13), p.10884
Main Authors: Torso, Nadine de Godoy, Quintanilha, Julia Coelho França, Cursino, Maria Aparecida, Pincinato, Eder de Carvalho, Lima, Carmen Silvia Passos, Moriel, Patricia
Format: Article
Language:English
Subjects:
Analysis
Cancer patients
Candidates
Care and treatment
Chemoradiotherapy
Chemotherapy
Cisplatin
Cisplatin - therapeutic use
Creatinine
Criteria
Efficiency
endogenous normalizer
Gene expression
Gene Expression Profiling
Genes
Germany
Head & neck cancer
Head and neck cancer
Head and Neck Neoplasms - drug therapy
Head and Neck Neoplasms - genetics
Humans
Kidneys
MicroRNA
MicroRNAs
MicroRNAs - genetics
MicroRNAs - metabolism
miRNA
qRT-PCR
reference gene
RNA sequencing
Urine
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Summary:The microRNA (miRNA) expression profile by qRT-PCR depends directly on the most appropriate normalization strategy adopted; however, currently there is no universally adequate reference gene. Therefore, this study aimed to determine, considering RNA-Seq results, the most adequate endogenous normalizer for use in the relative quantification of urine miRNAs from head and neck cancer patients, treated with cisplatin chemoradiotherapy. The massive sequencing was performed to identify the miRNAs differentially expressed between the group with cisplatin nephrotoxicity ( = 6) and the one without ( = 6). The candidate endogen normalizer was chosen according to four criteria: (1) the miRNA must be expressed in most samples; (2) the miRNA must have a fold change value between 0.99 and 1.01; (3) the miRNA must have a -value ≥ 0.98; and (4) the miRNA must not be commented on by the final GeneGlobe (Qiagen, Hilden, Germany) analysis. Four miRNAs met all the criteria (hsa-miR-363-5p, hsa-miR-875-5p, hsa-miR-4302, and hsa-miR-6749-5p) and were selected for validation by qRT-PCR in a cohort of 49 patients (including the 12 sequencing participants). Only hsa-miR-875-5p was shown to be an adequate normalizer for the experimental condition under investigation, as it exhibited invariant expression between the two groups.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms241310884