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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.


Sequence amplification and detection methods for small RNA ISH include (A) Padlock probes with RCA; (B) circular probes with RCA; (C) TIRCA; (D) O-FISH and (E) the ultramer extension method; Different methods for detection are also used for small RNAs including (F) direct labeling of probes (e.g., Cy3/fluorescein labeling); (G) NBT/BCIP; (H) TSA; (I) ELF and (J) silica spheres with Ru(bpy)32+. miRNA is presented as red line and probe is shown as black line. Violet dot represents non-fluorescent ligand.
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ijms-16-13259-f003: Sequence amplification and detection methods for small RNA ISH include (A) Padlock probes with RCA; (B) circular probes with RCA; (C) TIRCA; (D) O-FISH and (E) the ultramer extension method; Different methods for detection are also used for small RNAs including (F) direct labeling of probes (e.g., Cy3/fluorescein labeling); (G) NBT/BCIP; (H) TSA; (I) ELF and (J) silica spheres with Ru(bpy)32+. miRNA is presented as red line and probe is shown as black line. Violet dot represents non-fluorescent ligand.

Mentions: Several types of probes are combined with sequence amplification techniques to increase signal strength obtained from single miRNA molecules (Figure 3A–E). One example that enables sequence amplification is the use of padlock probes. These probes are successfully used to detect not only miRNAs but also mRNAs and DNA sequences. Padlock probes guarantee high specificity and sensitivity with single nucleotide discrimination [43]—what makes them applicable for allele-specific FISH [60]. Briefly, linear DNA probe after annealing to the specific sequence with 5′ and 3′ arms is circularized by DNA ligase. Circularization enables further signal amplification by rolling circle amplification (RCA) (Figure 3A). RCA uses miRNA molecule as a primer and elongates the sequence using circular probe as a template. Detection of RCA product is possible with the use of probes complementary to the sequence amplified on the template of the padlock probe central sequence. Similar types of probes, which are used in miRNA ISH also in combination with RCA, are circular DNA probes (Figure 3B). Circular probes are obtained in vitro with the use of padlock probes, ligation probes, and DNA ligase, and are then hybridized to the target sequence in cells as circular molecules [45].


Small RNA Detection by in Situ Hybridization Methods.

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

Sequence amplification and detection methods for small RNA ISH include (A) Padlock probes with RCA; (B) circular probes with RCA; (C) TIRCA; (D) O-FISH and (E) the ultramer extension method; Different methods for detection are also used for small RNAs including (F) direct labeling of probes (e.g., Cy3/fluorescein labeling); (G) NBT/BCIP; (H) TSA; (I) ELF and (J) silica spheres with Ru(bpy)32+. miRNA is presented as red line and probe is shown as black line. Violet dot represents non-fluorescent ligand.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-13259-f003: Sequence amplification and detection methods for small RNA ISH include (A) Padlock probes with RCA; (B) circular probes with RCA; (C) TIRCA; (D) O-FISH and (E) the ultramer extension method; Different methods for detection are also used for small RNAs including (F) direct labeling of probes (e.g., Cy3/fluorescein labeling); (G) NBT/BCIP; (H) TSA; (I) ELF and (J) silica spheres with Ru(bpy)32+. miRNA is presented as red line and probe is shown as black line. Violet dot represents non-fluorescent ligand.
Mentions: Several types of probes are combined with sequence amplification techniques to increase signal strength obtained from single miRNA molecules (Figure 3A–E). One example that enables sequence amplification is the use of padlock probes. These probes are successfully used to detect not only miRNAs but also mRNAs and DNA sequences. Padlock probes guarantee high specificity and sensitivity with single nucleotide discrimination [43]—what makes them applicable for allele-specific FISH [60]. Briefly, linear DNA probe after annealing to the specific sequence with 5′ and 3′ arms is circularized by DNA ligase. Circularization enables further signal amplification by rolling circle amplification (RCA) (Figure 3A). RCA uses miRNA molecule as a primer and elongates the sequence using circular probe as a template. Detection of RCA product is possible with the use of probes complementary to the sequence amplified on the template of the padlock probe central sequence. Similar types of probes, which are used in miRNA ISH also in combination with RCA, are circular DNA probes (Figure 3B). Circular probes are obtained in vitro with the use of padlock probes, ligation probes, and DNA ligase, and are then hybridized to the target sequence in cells as circular molecules [45].

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.