RNU6B, a frequent reference in miRNA expression studies, differentiates between deaths caused by hypothermia and chronic cardiac ischemia

Kaija, Helena; Pakanen, Lasse; Porvari, Katja

doi:10.1007/s00414-019-02041-0

Cited by 10 publications

(6 citation statements)

References 21 publications

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“…The expression of reference gene candidates in the pilot cohort. Two commonly used reference genes, U6 and SNORD-68 27,28,32 were selected as normalizers for the discovery of circulating biomarkers. We also included some other sncRNAs that have never been tested before as candidate biomarkers, such as miR-1275, miR-1280 and SNORD116.…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies, frequently used U6 sncRNA in miRNAs analysis profile, particularly in HCC 26,27 . However, various reports showed its instability across different sample sets 26,[28][29][30] . Several studies reported contradictory results about the comparison with an other miRNA as a reference gene for the normalization of the circulating miRNA expression in cancer setting [38][39][40] .…”

Section: Discussionmentioning

confidence: 99%

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Selection and validation of miR-1280 as a suitable endogenous normalizer for qRT-PCR Analysis of serum microRNA expression in Hepatocellular Carcinoma

Pratama

Cavalletto

Tiribelli

et al. 2020

Sci Rep

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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.Hepatocellular carcinoma (HCC) is one of the most common types of malignancies worldwide 1 . It is primarily known as the fourth-most common cause of mortality from cancer, contributing to 781.631 death in 2018 1 . Late diagnosis and the limited therapeutic approaches represent the ongoing challenges in liver oncology, responsible for the poor survival in patients 2-4 . Currently, numerous translational studies have been focused on the identification of circulatory biomarkers related to early diagnosis and prognosis 5,6 .Since their discovery in 1993, microRNAs (miRNAs) have been considered promising non-invasive biomarkers with wide application in many clinical settings 7,8 . MiRNAs are fundamental regulators of many cellular processes 9,10 and their alteration contributes to the disease onset, maintenance, and progression 11 . It has been demonstrated that miRNAs present in a stable cell-free form in the blood are not affected by temperatures, pH, technical handling, and storages. Different miRNAs profiles in blood circulation have been linked to cancer, including HCC, emphasizing the broad potential of miRNAs as a diagnostic and prognostic biomarkers 12-14 .One of the most popular technique to study the expression of miRNome in a biological sample is the quantitative reverse transcription polymerase chain reaction (qRT-PCR), which is widely preferred due to significant advantages such as sensitivity, large dynamic range, accuracy, and the convenient requirement of starting materials 15,16 . Standardization and normalization procedures are sometimes recommended for the comparison of miRNA expression between different samples to reduce the variability caused by pre-analytical factors such as specimen collection, p...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Selection and validation of miR-1280 as a suitable endogenous normalizer for qRT-PCR Analysis of serum microRNA expression in Hepatocellular Carcinoma

Pratama

Cavalletto

Tiribelli

et al. 2020

Sci Rep

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“…Postmortem biochemistry is increasingly used in medico-legal death investigations, and more recently also molecular pathology. [ 13 ] Our recent finding concerning microRNA U6B small nuclear RNA (RNU6B) expression [ 14 ] could be useful in forensic molecular diagnostics.…”

Section: Discussionmentioning

confidence: 99%

Low myocardial transcript variant alt-a of cyclin dependent kinase inhibitor p21 expression differentiates hypothermia from cardiac/respiratory causes of death

2020

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Gene expressions in the myocardium have been shown to vary between different causes of death, which can be utilized in the recognition of varied processes. Our previous work with a limited number of cases showed a high messenger ribonucleic acid expression of the transcript variant alt-a of cyclin dependent kinase inhibitor p21 ( p21 alt-a ) in chronic cardiac ischemia deaths and a low expression in hypothermia deaths and acute myocardial ischemia deaths. In present work, p21 alt-a expression in the myocardium of human cadavers was calculated using glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) as reference gene. In this collection of 143 samples, the p21 alt-a expression was significantly lower in hypothermia than in chronic cardiac ischemic heart disease with ( P < .001) or without ( P < .001) acute myocardial infarction and in other cardiac and respiratory disease deaths ( P < .000). Chronic ischemic heart disease in hypothermia cases did not increase the expression. The p21 alt-a expression did not correlate with postmortem interval, quality of RNA or with the age of the deceased. The p21 alt-a referenced to GAPDH expression in cadaver myocardium has apparent potential as a marker distinguishing between hypothermia and cardiac/respiratory diseases as causes of death.

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“…Similarly, miR-NAs enriched (fold change > 1:4 and p ≤ 0:05) in MetS-EVs compared to MetS-MSCs were identified. For validation purposes, we randomly selected 4 miRNAs (2 upregulated and 2 downregulated in MetS-EVs versus Lean-EVs) and measured their expression by quantitative polymerase chain reaction (qPCR) using RNA U6 small nuclear 6 pseudogene (RNU6B) as normalization/internal control, a common reference gene in miRNA expression studies [30]. We then used TargetScan to identified genes target by these miRNAs and filtered these genes by MitoCarta [31] to identify mitochondria-related genes.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Syndrome Alters the Cargo of Mitochondria-Related microRNAs in Swine Mesenchymal Stem Cell-Derived Extracellular Vesicles, Impairing Their Capacity to Repair the Stenotic Kidney

Farahani

Zhu

Tang

et al. 2020

Stem Cells International

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Background. Coexisting metabolic syndrome (MetS) and renal artery stenosis (RAS) are linked to poor renal outcomes. Mesenchymal stem/stromal cell- (MSC-) derived extracellular vesicles (EVs) from lean animals show superior ability to repair the experimental MetS+RAS kidney compared to EVs from MetS pig MSCs. We hypothesized that MetS leads to selective packaging in porcine EVs of microRNAs capable of targeting mitochondrial genes, interfering with their capacity to repair the MetS+RAS kidney. Methods. Five groups of pigs ( n = 7 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS+RAS) and MetS+RAS 4 weeks after a single intrarenal delivery of EVs harvested from allogeneic adipose tissue-derived MSCs isolated from Lean or MetS pigs, and Lean or MetS sham controls. Single-kidney blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector CT, whereas EV microRNA cargo, renal tubular mitochondrial structure and bioenergetics, and renal injury pathways were assessed ex vivo. Results. microRNA sequencing revealed 19 dysregulated microRNAs capable of targeting several mitochondrial genes in MetS-EVs versus Lean-EVs. Lean- and MetS-EVs were detected in the stenotic kidney 4 weeks after administration. However, only MetS-EVs failed to improve renal mitochondrial density, structure, and function or attenuate oxidative stress, tubular injury, and fibrosis. Furthermore, Lean-EVs but not MetS-EVs restored RBF and GFR in MetS+RAS. Conclusion. MetS alters the cargo of mitochondria-related microRNAs in swine MSC-derived EVs, which might impair their capacity to repair the poststenotic kidney in MetS+RAS. These observations may contribute to develop approaches to improve the efficacy of MSC-EVs for patients with MetS.

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RNU6B, a frequent reference in miRNA expression studies, differentiates between deaths caused by hypothermia and chronic cardiac ischemia

Cited by 10 publications

References 21 publications

Selection and validation of miR-1280 as a suitable endogenous normalizer for qRT-PCR Analysis of serum microRNA expression in Hepatocellular Carcinoma

Selection and validation of miR-1280 as a suitable endogenous normalizer for qRT-PCR Analysis of serum microRNA expression in Hepatocellular Carcinoma

Low myocardial transcript variant alt-a of cyclin dependent kinase inhibitor p21 expression differentiates hypothermia from cardiac/respiratory causes of death

Metabolic Syndrome Alters the Cargo of Mitochondria-Related microRNAs in Swine Mesenchymal Stem Cell-Derived Extracellular Vesicles, Impairing Their Capacity to Repair the Stenotic Kidney

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