Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

Egidi, Maria Giulia; Cochetti, Giovanni; Guelfi, Gabriella; Zampini, Danilo; Diverio, Silvana; Poli, Giulia; Mearini, Ettore

doi:10.1155/2015/973597

Cited by 36 publications

(38 citation statements)

References 44 publications

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“…It has been demonstrated that miRNAs are detectable in urine of PCa patients collected after DRE (digital rectal examination) 13 , 14 . As demonstrated in our last study quantitative real-time PCR (qPCR) miRNA analysis could be considered a non-invasive promising tool for diagnostic testing on PCa, and on its prognostic status 15 .…”

Section: Introductionmentioning

confidence: 99%

Next Generation Sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research

Guelfi

Cochetti

Stefanetti

et al. 2018

Sci Rep

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There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.

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Section: Introductionmentioning

confidence: 99%

Next Generation Sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research

Guelfi

Cochetti

Stefanetti

et al. 2018

Sci Rep

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“…The progressive confirmation of their key role in cancer, their stability in biological fluids and their resistance to various storage conditions6–9 make miRNAs excellent candidates for the development of minimally invasive biomarkers for cancer diagnosis and prognosis. Unfortunately, a universally accepted procedure to deal with miRNAs does not exist, and data normalization is currently performed through the experimental assessment of candidate housekeeping genes 10. This study was aimed at characterizing a panel of circulating miRNAs that could distinguish PCa from benign prostatic hyperplasia (BPH) in a population of age-matched patients with increased PSA levels.…”

Section: Introductionmentioning

confidence: 99%

Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role

Cochetti¹,

Poli²,

Guelfi³

et al. 2016

OTT

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IntroductionDiagnosis of prostate cancer (PCa) is based on prostate biopsy that is performed when prostate specific antigen (PSA) is persistently altered over time and/or abnormal digital rectal examination is found. Serum PSA levels increase in both PCa and benign prostatic hyperplasia, leading to an increased number of unnecessary biopsies. There is an urgent need to unravel PCa-specific molecular signatures.Patients and methodsThis study aimed at characterizing a panel of circulating micro RNAs (miRNAs) that could distinguish PCa from benign prostatic hyperplasia in a population of age-matched patients with increased PSA levels. Both miRNAs targeting genes involved in PCa onset and miRNAs whose role in PCa has been highlighted in other studies were included. For this purpose, let-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-24-3p, miR-23b-3p, miR-27-b-3p, miR-106a-5p, miR-20b-5p, miR-18b-5p, miR-19b-2-5p, miR-363-3p, miR-497, miR-195, miR-25-3p, miR-30c-5p, miR-622, miR-874-3p, miR-346 and miR-940 were assayed through real-time PCR in 64 patients with PCa and compared with 60 patients with benign prostatic hyperplasia. The ability of miRNAs to predict the stage of disease was also analyzed.ResultsLet-7c, let-7e, let-7i, miR-26a-5p, miR-26b-5p, miR-18b-5p and miR-25-3p were able to discriminate patients with PCa from those harboring benign prostatic hyperplasia, both presenting altered PSA levels (>3 ng/mL). MiR-25-3p and miR-18b-5p showed the highest sensitivity and specificity to predict PCa, respectively. The combination of these two miRNAs improved the overall sensitivity. A correlation between pathological Gleason score and miRNA expression levels was reported; miR-363-3p, miR-26a-5p, miR-26b-5p, miR-106a-5p, miR-18b-5p, miR-25-3p and let-7i decreased in expression concomitantly with an increase in malignancy.ConclusionThis study confirms serum miRNAs to be reliable candidates for the development of minimally invasive biomarkers for the diagnosis and prognosis of PCa, particularly in those cases where PSA acts as a flawed marker.

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“…They also cause changes in gene expression, which can be accurately and sensitively measured by qRT-PCR [17, 20]. This technique normalizes the gene of interest against an endogenous control whose expression remains unaltered in the samples under analysis [21]. The concept of validating reference genes used for normalization in qRT-PCR analysis before use was initially suggested in 2002 [22], and has been realized in various scientific disciplines such as plant sciences [23, 24], cancer [25, 26], stem cells [27, 28], and cardiovascular research [14, 29, 30].…”

Section: Discussionmentioning

confidence: 99%

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Diao

Zhao

et al. 2017

BMC Molecular Biol

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BackgroundOxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).ResultsUsing geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

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Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

Cited by 36 publications

References 44 publications

Next Generation Sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research

Next Generation Sequencing of urine exfoliated cells: an approach of prostate cancer microRNAs research

Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

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