miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses

Smoczynska, Aleksandra; Sega, Pawel; Stępień, Agata; Knop, Katarzyna; Jarmołowski, Artur; Pacak, Andrzej; Szweykowska-Kulińska, Zofia

doi:10.1007/978-1-4939-9042-9_10

Cited by 13 publications

(14 citation statements)

References 25 publications

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“…This method involves the separation of the total amount of RNA on polyacrylamide gel that possesses the property of denaturation, followed by its transfer on a nylon membrane. After that, the RNA undergoes a process of UV cross-linking and, lastly, it is hybridized using a radioactive substance [96][97][98][99]. However, this technique tends to be strenuous, it necessitates large quantities of RNA and it has sometimes been reported to omit rare types of miRNA [99].…”

Section: Mirna Identification Techniquesmentioning

confidence: 99%

“…After that, the RNA undergoes a process of UV cross-linking and, lastly, it is hybridized using a radioactive substance [96][97][98][99]. However, this technique tends to be strenuous, it necessitates large quantities of RNA and it has sometimes been reported to omit rare types of miRNA [99]. Under these circumstances, efforts have been made in order to improve the method, therefore leading to the possibility of using lower amounts of RNA and also shortening the execution time of the technique [100][101][102].…”

Section: Mirna Identification Techniquesmentioning

confidence: 99%

“…The second method is next-generation sequencing or NGS. Despite the fact that this is a highly accurate technique that has the ability to detect single miRNAs with the precision of one nucleotide, its high costs lead to a limitation of this technique's wider accessibility [99].…”

Section: Mirna Identification Techniquesmentioning

confidence: 99%

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miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis

Condrat

Thompson

Barbu

et al. 2020

Cells

925

583

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MicroRNAs (miRNAs) represent a class of small, non-coding RNAs with the main roles of regulating mRNA through its degradation and adjusting protein levels. In recent years, extraordinary progress has been made in terms of identifying the origin and exact functions of miRNA, focusing on their potential use in both the research and the clinical field. This review aims at improving the current understanding of these molecules and their applicability in the medical field. A thorough analysis of the literature consulting resources available in online databases such as NCBI, PubMed, Medline, ScienceDirect, and UpToDate was performed. There is promising evidence that in spite of the lack of standardized protocols regarding the use of miRNAs in current clinical practice, they constitute a reliable tool for future use. These molecules meet most of the required criteria for being an ideal biomarker, such as accessibility, high specificity, and sensitivity. Despite present limitations, miRNAs as biomarkers for various conditions remain an impressive research field. As current techniques evolve, we anticipate that miRNAs will become a routine approach in the development of personalized patient profiles, thus permitting more specific therapeutic interventions.Biogenesis of miRNA begins in the nucleus, where the transcription of its precursor, primary miRNA or pri-miRNA takes place under the influence of RNA polymerases II and III [11,12]. The resulting molecule is a hairpin-like structure, which contains a loop at one end [11]. This primordial mi-RNA precursor that is usually made up of hundreds of nucleotides is then processed consecutively by two RNase III enzymes [13][14][15]. The first enzyme to act upon the pri-miRNA, which still resides in the nucleus, is called Drosha or DCGR8, and turns it into a new hairpin-like structure of approximately 70 nucleotides, the Precursor-miRNA or pre-miRNA. The latter is then transported to the cytoplasm, with the help of Exportin-5, where it is cleaved again by the Ago2/Dicer complex leading to the short, mature miRNA double strands [16].Further on, one of the strands, usually known as the guide strand, will be integrated into the RNA-induced silencing complex (RISC), while the other one, known as the passenger strand, is going to be degraded, even though in some occasions it has been found to be also functional [17]. In most cases, the strand that contains the less stable 5 end or a uracil at the beginning is more likely to be selected as the guide strand [18][19][20]. In those situations, where the passenger strand is not degraded and both get incorporated into the miRISC complex, the mature miRNA in the guide strand will be the dominant one [21,22].The main role of miRNA in the human body is gene regulation [23] by mediating the degradation of mRNA and also by regulating transcription and translation through canonical and non-canonical mechanisms [4]. The canonical mechanism means that the miRISC complex containing the miRNA guide strand is exerting its action by binding to the targ...

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Section: Mirna Identification Techniquesmentioning

confidence: 99%

Section: Mirna Identification Techniquesmentioning

confidence: 99%

See 1 more Smart Citation

miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis

Condrat

Thompson

Barbu

et al. 2020

Cells

925

583

View full text Add to dashboard Cite

show abstract

“…Another popular quantitative method for the detection of miRNAs is Northern blot hybridization, which requires the total RNA to be separated on polyacrylamide gel with denaturation proprieties, after which it is transferred to a nylon membrane, UV-cross-linked and finally hybridized with the help of a probe labeled with a radioactive substance (Kruszka et al, 2014;Barciszewska-Pacak et al, 2015;Pacak et al, 2016;Smoczynska et al, 2019). Still, it is a laborious, time-consuming technique which requires high amounts of RNA and can sometimes miss the identification of rare miRNAs (Smoczynska et al, 2019). Recently, new protocols have been developed in order to enhance the technique, leading to the use of lower levels of RNA and to a shortening of the time needed to execute the procedure (Varallyay et al, 2007;Pall and Hamilton, 2008;.…”

Section: Proviral Micrornasmentioning

confidence: 99%

“…The general principle on which they all function is based on the amplification and sequencing of DNA fragments, with additional steps for miRNA sequencing, starting with miRNA extraction after which the miRNAs are reverse transcribed into cDNA (Bar et al, 2008;Creighton et al, 2009;Hu et al, 2017). NGS is able to identify single miRNA with the high resolution of one nucleotide, yet the Pichiorri et al, 2008;Kalluri and Weinberg, 2009;Jiang et al, 2010;Chen Y. et al, 2011;Su et al, 2011;Jopling, 2012;Wang et al, 2012;Xu et al, 2012;Yao et al, 2012;Li et al, 2013 - high cost of this method, compared to the others, limits its accessibility (Smoczynska et al, 2019). However, this technique has numerous other crucial attributes that should be taken into consideration when choosing a quantification method, such as its high throughout, meaning that the samples one researcher sends would be sequenced in the same time with many other samples (Hu et al, 2017).…”

Section: Proviral Micrornasmentioning

confidence: 99%