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Methods for quantification of microRNA and small interfering RNA

A polymerization, anchoring nucleotide technique, used in the field of detection and quantification of target DNA sequences, which can solve the problem that the factors of rate are poorly understood and none of them are sufficient

Active Publication Date: 2007-05-09
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently, little is known about the rates at which alternative splicing patterns or RNA editing change, and the factors that influence these rates
[0029] Previously, other assays were performed to detect splicing of RNA transcripts in yeast itself, or putative exon skipping splicing events in rat tissues, but none of these methods had sufficient resolution. rate to estimate the amount of splice variants that may be necessary for understanding alterations in the cell life cycle and in disease

Method used

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  • Methods for quantification of microRNA and small interfering RNA
  • Methods for quantification of microRNA and small interfering RNA
  • Methods for quantification of microRNA and small interfering RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0331] Real-time quantitative PCR assay for human miR-15a microRNA target sequence

[0332] Sequence-specific LNA-modified microRNA tagging probes are annealed and ligated. The ligated template was subsequently detected with real-time PCR, anchor PCR primers for miR-15a microRNA, and an LNA-modified dual-labeled detection probe, and the negative template was used as a negative control. The specificity of this reaction was tested using a reaction without ligase. The cycle threshold (Ct), which represents the PCR cycle at which the increase in reporter fluorescence above the baseline signal can be detected for the first time, was 35.0 for the attached microRNA probe using the miR-15a microRNA template (Fig. 2A), While no Ct was observed for negative control experiments (negative template and negative ligase, respectively). A normalized reporter signal (Rn), which represents the fluorescent signal of the reporter dye divided by the fluorescent signal of the passive reference dy...

Embodiment 2

[0334] Real-time quantitative PCR assay for human miR-15a microRNA target sequence and corresponding DNA 3'-block target

[0335] The RNA template was replaced with a DNA template chemically blocked with a phosphate at the 3'-end. Without the addition of ligase in the ligation reaction, the blocked DNA template cannot be detected in the LNA sequence-specific real-time PCR assay. The Ct values ​​of RNA template and DNA template were 35.0 and 33.3, respectively (Fig.3).

Embodiment 3

[0337] Specificity of real-time quantitative PCR assays for human miR-15a and human miR-16 microRNA target sequences

[0338] The sequence-specific microRNA target sequence recognition of the present invention was assessed by using the miR-15a microRNA target compared to the human miR-16 target, which has 72% sequence identity to the miR-15a target sequence. Neither the negative template control nor the no template control (NTC) showed any signal in the real-time PCR reaction. Using the hybridization conditions used to anneal the LNA-modified miR-15a target sequence-specific tagging probe to the miR-15a target as described above resulted in a Ct value of 36.2, while using the same tagging probe for the highly homologous The specific miR-16 obtained a Ct value of 39.9, corresponding to a 13-fold discriminative difference (Fig. 4).

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Abstract

The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to method s for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification a s well as monitoring the expression of deoxy nucleic acids.

Description

[0001] The present invention relates to nucleic acids, probes and methods for detecting, quantifying and monitoring the expression of mature microRNAs (microRNAs) and small interfering RNAs (siRNAs). The present invention further relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, and detection and quantification of RNA edits, allelic variants of single transcripts, mutations, deletions or duplications of specific exons within a transcript, For example methods of alterations associated with human diseases such as cancer. The invention furthermore relates to methods for the detection and quantification of target DNA sequences. Background of the invention [0002] The present invention relates to the quantification of target nucleotide sequences in a variety of nucleic acid samples and more particularly to methods employing the design and use of oligonucleotide probes that can be used to detect and quantify target nucleotides of inter...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6853C12Q1/6851C12Q2525/161C12Q2525/155C12Q2521/107
Inventor N·雅各布森L·孔斯巴克S·考皮南S·M·埃克瓦尔德P·莫里森P·S·尼尔森M·诺霍尔姆
Owner QIAGEN GMBH
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