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Small RNA Detection by in Situ Hybridization Methods.

Urbanek MO, Nawrocka AU, Krzyzosiak WJ - Int J Mol Sci (2015)

Bottom Line: In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection.We discuss relevant technical aspects as well as the advantages and limitations of ISH.We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland. martyna.urbanek@ibch.poznan.pl.

ABSTRACT
Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

No MeSH data available.


In situ hybridization protocols used for imaging of small RNAs. On the left are the steps of the ISH protocol on a cellular/tissue level, in the center are steps on a molecular level and on the right are modification variants of the ISH protocol used to detect small RNAs. Red and green dots represent fluorophores and squares represent non-fluorescent ligands. Black crosses indicate immobilization of miRNA.
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ijms-16-13259-f001: In situ hybridization protocols used for imaging of small RNAs. On the left are the steps of the ISH protocol on a cellular/tissue level, in the center are steps on a molecular level and on the right are modification variants of the ISH protocol used to detect small RNAs. Red and green dots represent fluorophores and squares represent non-fluorescent ligands. Black crosses indicate immobilization of miRNA.

Mentions: miRNA ISH is exceptionally challenging because of miRNA features such as small size, sequence similarity among various miRNA family members and low tissue-specific or development-specific expression levels. The standard ISH protocol was modified to improve miRNA detection (Figure 1, Table 1) in various types of cell lines and tissues as well as whole embryos. Here, we describe these modifications with a focus on technical aspects and critically discuss these adaptations in the context of single molecule ISH and multi-miRNA detection.


Small RNA Detection by in Situ Hybridization Methods.

Urbanek MO, Nawrocka AU, Krzyzosiak WJ - Int J Mol Sci (2015)

In situ hybridization protocols used for imaging of small RNAs. On the left are the steps of the ISH protocol on a cellular/tissue level, in the center are steps on a molecular level and on the right are modification variants of the ISH protocol used to detect small RNAs. Red and green dots represent fluorophores and squares represent non-fluorescent ligands. Black crosses indicate immobilization of miRNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490494&req=5

ijms-16-13259-f001: In situ hybridization protocols used for imaging of small RNAs. On the left are the steps of the ISH protocol on a cellular/tissue level, in the center are steps on a molecular level and on the right are modification variants of the ISH protocol used to detect small RNAs. Red and green dots represent fluorophores and squares represent non-fluorescent ligands. Black crosses indicate immobilization of miRNA.
Mentions: miRNA ISH is exceptionally challenging because of miRNA features such as small size, sequence similarity among various miRNA family members and low tissue-specific or development-specific expression levels. The standard ISH protocol was modified to improve miRNA detection (Figure 1, Table 1) in various types of cell lines and tissues as well as whole embryos. Here, we describe these modifications with a focus on technical aspects and critically discuss these adaptations in the context of single molecule ISH and multi-miRNA detection.

Bottom Line: In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection.We discuss relevant technical aspects as well as the advantages and limitations of ISH.We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 Str., 61-704 Poznan, Poland. martyna.urbanek@ibch.poznan.pl.

ABSTRACT
Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

No MeSH data available.