very high specificity, without false positives, and is sensitive for low amounts of these
short RNA targets (such as 10 pg of total RNA). In the LNA assay, the RT-PCR
reaction is performed with LNA primers. LNA is a modified RNA nucleotide in
which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding.
LNA-enhanced oligonucleotides improve sensitivity and specificity of RT-PC,
increasing the affinity for its DNA or RNA complementary oligos, mainly for small
or highly similar targets. Thermal stability considerably enhances the Tm of the
duplex, which increases by 2–8
C. In addition, LNA primers are efficient at discriminating single-nucleotide mismatches. In LNA RT-PCR, miRNAs are reverse transcribed from total RNA using miRNA-specific RT primers; reverse-transcribed
miRNAs are then amplified using an LNA-enhanced PCR primer together with a
universal PCR primer (Fig. 6b).
LNA oligos are also used in hybridization-based technologies including qRT-PCR
platforms (e.g. Qiagen), microarray and in situ hybridization (ISH). For quantification of circulating miRNAs, new technologies such as droplet digital PCR (ddPCR),
Ligo-miR and Two-tailed RT-qPCR have recently been introduced to overcome
miRNA normalization in biofluids and to detect even single copies with high repeatability. The ddPCR method determines absolute miRNA expression and is based on
partitioning of the sample into thousands of discrete, volumetrically defined, wateroil emulsion droplets. PCR amplification of the template molecules occurs in each
individual droplet using reagents and workflows similar to those used for most
standard TaqMan probe-based assays. Following PCR, each droplet is analysed by
estimation of the number of molecules in the reaction under the assumption of a
Poisson distribution. Results are expressed as target copies per microlitre of reaction
[65, 66]. The high cost and complexity of ddPCR make it difficult to screen large
numbers of samples across large panels of miRNA. Ligo-miR is a multiple assay
technology to rapidly measure absolute miRNA copy numbers from a wide range of
biological sources. Ligo-miR assay comprises two sequential ligation steps: capture
and coding (Fig. 7). Firstly, a DNA universal adapter is ligated to the 3
0 end of all
miRNA molecules. Then in the coding ligation up to 26 miRNA-specific discrimination probes and an Alexa647-labelled common probe are hybridized to the miRNA
templates to form a single-stranded DNA product. Thermal cycling is used to perform
a 50X linear amplification by repeatedly generating Ligo-miR products from each
miRNA template. The products can be identified and quantified using common DNA
sizing methods such as electrophoresis (denaturing PAGE). Each band is a specific
miRNA product where band intensity is proportional to quantity. Absolute copy
numbers can be determined using the included spike-in controls and running reference samples. The sensitivity of Ligo-miR is as low as 20 copies per cell at 75 ng input
levels [67]. Two-tailed RT-qPCR is a very recent system for the quantification of
miRNAs (Fig. 8). In the RT step, two specific primers about 50 nucleotides long and
complementary to the miRNA target are used, while the cDNA is amplified by qPCR
using two target-specific primers and SYBR Green chemistry [68].
Advanced Assays in Epigenetics
547
short RNA targets (such as 10 pg of total RNA). In the LNA assay, the RT-PCR
reaction is performed with LNA primers. LNA is a modified RNA nucleotide in
which the ribose ring is “locked” in the ideal conformation for Watson-Crick binding.
LNA-enhanced oligonucleotides improve sensitivity and specificity of RT-PC,
increasing the affinity for its DNA or RNA complementary oligos, mainly for small
or highly similar targets. Thermal stability considerably enhances the Tm of the
duplex, which increases by 2–8
C. In addition, LNA primers are efficient at discriminating single-nucleotide mismatches. In LNA RT-PCR, miRNAs are reverse transcribed from total RNA using miRNA-specific RT primers; reverse-transcribed
miRNAs are then amplified using an LNA-enhanced PCR primer together with a
universal PCR primer (Fig. 6b).
LNA oligos are also used in hybridization-based technologies including qRT-PCR
platforms (e.g. Qiagen), microarray and in situ hybridization (ISH). For quantification of circulating miRNAs, new technologies such as droplet digital PCR (ddPCR),
Ligo-miR and Two-tailed RT-qPCR have recently been introduced to overcome
miRNA normalization in biofluids and to detect even single copies with high repeatability. The ddPCR method determines absolute miRNA expression and is based on
partitioning of the sample into thousands of discrete, volumetrically defined, wateroil emulsion droplets. PCR amplification of the template molecules occurs in each
individual droplet using reagents and workflows similar to those used for most
standard TaqMan probe-based assays. Following PCR, each droplet is analysed by
estimation of the number of molecules in the reaction under the assumption of a
Poisson distribution. Results are expressed as target copies per microlitre of reaction
[65, 66]. The high cost and complexity of ddPCR make it difficult to screen large
numbers of samples across large panels of miRNA. Ligo-miR is a multiple assay
technology to rapidly measure absolute miRNA copy numbers from a wide range of
biological sources. Ligo-miR assay comprises two sequential ligation steps: capture
and coding (Fig. 7). Firstly, a DNA universal adapter is ligated to the 3
0 end of all
miRNA molecules. Then in the coding ligation up to 26 miRNA-specific discrimination probes and an Alexa647-labelled common probe are hybridized to the miRNA
templates to form a single-stranded DNA product. Thermal cycling is used to perform
a 50X linear amplification by repeatedly generating Ligo-miR products from each
miRNA template. The products can be identified and quantified using common DNA
sizing methods such as electrophoresis (denaturing PAGE). Each band is a specific
miRNA product where band intensity is proportional to quantity. Absolute copy
numbers can be determined using the included spike-in controls and running reference samples. The sensitivity of Ligo-miR is as low as 20 copies per cell at 75 ng input
levels [67]. Two-tailed RT-qPCR is a very recent system for the quantification of
miRNAs (Fig. 8). In the RT step, two specific primers about 50 nucleotides long and
complementary to the miRNA target are used, while the cDNA is amplified by qPCR
using two target-specific primers and SYBR Green chemistry [68].
Advanced Assays in Epigenetics
547