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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.


Types of nucleotide modifications used in small RNA ISH probes. (A) Chemical structure of modified nucleotides present in the probes; (B) Comparison of the melting temperature (Tm) of the 22 and 16 nt probes with different modifications (marked in bold) and RNA target (hsa-let-7a-1). Target sequence for shorter probe is marked in italic. The high melting temperature indicates strong binding with the target sequence. All Tm calculations were performed with IDT Technologies OligoAnalyzer 3.1; (C) Frequency of different types of probes used in small RNA ISH.
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ijms-16-13259-f002: Types of nucleotide modifications used in small RNA ISH probes. (A) Chemical structure of modified nucleotides present in the probes; (B) Comparison of the melting temperature (Tm) of the 22 and 16 nt probes with different modifications (marked in bold) and RNA target (hsa-let-7a-1). Target sequence for shorter probe is marked in italic. The high melting temperature indicates strong binding with the target sequence. All Tm calculations were performed with IDT Technologies OligoAnalyzer 3.1; (C) Frequency of different types of probes used in small RNA ISH.

Mentions: In standard ISH, probes composed of DNA or RNA nucleotides are commonly used. Unmodified DNA and RNA probes have relatively poor binding affinity to target sequences [54], and therefore several modifications have been proposed to improve their properties (Figure 2). First and most commonly applied was Locked Nucleic Acid (LNA) modification, which remains the gold standard in RNA FISH not only in small RNA detection. LNA nucleotides, referred to as “locked” RNA, have an additional bridge connecting 4′C and 2′O atoms. LNA nucleotides are incorporated into DNA probes, which leads to the formation of hybrid LNA/DNA probes. LNA/DNA probes have been shown to be highly beneficial in miRNA detection because of a short hybridization time, high efficiency, discriminatory power and a high melting temperature of the miRNA:probe complex. The minimal length of the LNA/DNA probe was determined to be 12 nucleotides [55] and these probes usually contain 30% LNA nucleotides. Besides their unquestionable advantages, these probes are expensive and can generate strong background signals resulting in a low signal-to-noise ratio for low abundant miRNA [56]. Therefore, other modifications were also proposed. These modifications include 2′fluoro-modified RNA (2′F RNA), morpholino, Zip Nucleic Acids (ZNA) [57], N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) [58] and 2′O-Methyl (2′OMe) RNA modification. In comparison to DNA probes, 2′OMe RNA probes have faster hybridization kinetics and the ability to bind structured targets under standard conditions [59]. The combination of 2′OMe RNA and LNA modifications (in a 2:1 ratio) resulted in improved specificity and stability of the probe:RNA duplex in comparison to the LNA/DNA probe [38]. Specificity of the system may be further improved by shortening the probe length to 19 nt [59]. As the LNA/2′OMe RNA probe binds more strongly to yeast RNA or salmon sperm RNA used in ISH as a standard blocking agent, better results were obtained without these RNA blockers in the hybridization step [38]. 2′F RNA nucleotides incorporated in the DNA probes ensure increased binding to the target and better nuclease resistance [41]. Morpholino modifications, often applied to inhibit translation, modify splicing patterns of the primary transcript, or block miRNAs, were also used to detect miRNAs because of their high stability [42].


Small RNA Detection by in Situ Hybridization Methods.

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

Types of nucleotide modifications used in small RNA ISH probes. (A) Chemical structure of modified nucleotides present in the probes; (B) Comparison of the melting temperature (Tm) of the 22 and 16 nt probes with different modifications (marked in bold) and RNA target (hsa-let-7a-1). Target sequence for shorter probe is marked in italic. The high melting temperature indicates strong binding with the target sequence. All Tm calculations were performed with IDT Technologies OligoAnalyzer 3.1; (C) Frequency of different types of probes used in small RNA ISH.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-13259-f002: Types of nucleotide modifications used in small RNA ISH probes. (A) Chemical structure of modified nucleotides present in the probes; (B) Comparison of the melting temperature (Tm) of the 22 and 16 nt probes with different modifications (marked in bold) and RNA target (hsa-let-7a-1). Target sequence for shorter probe is marked in italic. The high melting temperature indicates strong binding with the target sequence. All Tm calculations were performed with IDT Technologies OligoAnalyzer 3.1; (C) Frequency of different types of probes used in small RNA ISH.
Mentions: In standard ISH, probes composed of DNA or RNA nucleotides are commonly used. Unmodified DNA and RNA probes have relatively poor binding affinity to target sequences [54], and therefore several modifications have been proposed to improve their properties (Figure 2). First and most commonly applied was Locked Nucleic Acid (LNA) modification, which remains the gold standard in RNA FISH not only in small RNA detection. LNA nucleotides, referred to as “locked” RNA, have an additional bridge connecting 4′C and 2′O atoms. LNA nucleotides are incorporated into DNA probes, which leads to the formation of hybrid LNA/DNA probes. LNA/DNA probes have been shown to be highly beneficial in miRNA detection because of a short hybridization time, high efficiency, discriminatory power and a high melting temperature of the miRNA:probe complex. The minimal length of the LNA/DNA probe was determined to be 12 nucleotides [55] and these probes usually contain 30% LNA nucleotides. Besides their unquestionable advantages, these probes are expensive and can generate strong background signals resulting in a low signal-to-noise ratio for low abundant miRNA [56]. Therefore, other modifications were also proposed. These modifications include 2′fluoro-modified RNA (2′F RNA), morpholino, Zip Nucleic Acids (ZNA) [57], N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (ZEN) [58] and 2′O-Methyl (2′OMe) RNA modification. In comparison to DNA probes, 2′OMe RNA probes have faster hybridization kinetics and the ability to bind structured targets under standard conditions [59]. The combination of 2′OMe RNA and LNA modifications (in a 2:1 ratio) resulted in improved specificity and stability of the probe:RNA duplex in comparison to the LNA/DNA probe [38]. Specificity of the system may be further improved by shortening the probe length to 19 nt [59]. As the LNA/2′OMe RNA probe binds more strongly to yeast RNA or salmon sperm RNA used in ISH as a standard blocking agent, better results were obtained without these RNA blockers in the hybridization step [38]. 2′F RNA nucleotides incorporated in the DNA probes ensure increased binding to the target and better nuclease resistance [41]. Morpholino modifications, often applied to inhibit translation, modify splicing patterns of the primary transcript, or block miRNAs, were also used to detect miRNAs because of their high stability [42].

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.