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RNA in situ hybridization

An in situ hybridization and oligonucleotide technology, applied in the field of RNA in situ hybridization, can solve the problems of increasing background noise and high permeability of tissue samples, and achieve the effect of improving diagnostic accuracy

Inactive Publication Date: 2011-04-27
CELISH FD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, even if fish sperm DNA is fragmented to a length of about 2000 bases, the oligonucleotide probe is shorter than fish sperm DNA, and its permeability to tissue samples is high, so there may be fish sperm in tissue samples Non-specific adsorption sites that DNA cannot reach, which may also contribute to increased background noise

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0153] Embodiment 1 (the experiment of false oligonucleotide addition ratio)

[0154] The optimum addition ratio corresponding to the probe concentration of the dummy oligo DNA was obtained. Mouse liver was used as the target tissue, and the gene to be detected was Cyp1a2. The livers of 8-week-old male mice were usually fixed in formalin and embedded in paraffin to make paraffin blocks, made into serial sections with a thickness of 5 μm, and deparaffinized, and then proteinase K (Invitrogen Company, Proteinase K SOL.RNA, 25530049) treatment, followed by RNA in situ hybridization. As probes for detecting mRNA of the gene Cyp1a2, four types of single-stranded oligo DNA probes (SEQ ID NOS: 1 to 4) whose both ends were labeled with FITC were used. On the mRNA of the Cyp1a2 gene, sequence numbers 1 to 4 are arranged in the order of numbering along the direction from the 5' end to the 3' end, and the distance to the adjacent oligo DNA probe is 594 for sequence numbers 1 and 2 Bas...

Embodiment 2

[0155] Embodiment 2 (false oligomeric DNA, salmon sperm DNA and the comparison without adding)

[0156] The effects of pseudo-oligo DNA, salmon sperm DNA, and quantitative RNA in situ hybridization RNA detection without addition were compared. In the experiment, a formalin-fixed and paraffin-embedded mouse liver tissue sample was used in the same manner as in Example 1, and serial sections were prepared and used in the experiment. The detection gene is the same as in Example 1, using Cyp1a2 and FITC-labeled 4 kinds of single-stranded oligomeric DNA probes shown in sequence numbers 1 to 4 at both ends, and the respective concentrations of the 4 kinds of probes are 0 nM (nano mol), 1nM, 2nM, 3nM, 4nM, and 5nM, corresponding to various probe concentrations, and the concentration shown in SEQ ID NO: 5 (denoted as L1C1) and SEQ ID NO: 6 (denoted as arbp) was used at a concentration that was 8 times the addition ratio. 2 types of pseudo-oligomeric DNA (single-stranded). In additio...

Embodiment 3

[0157] Embodiment 3 (comparison of false oligomeric DNA and salmon sperm DNA)

[0158]The effects of quantitative RNA in situ hybridization on false oligo DNA and salmon sperm DNA in RNA detection were compared. In the experiment, serial sections were prepared from formalin-fixed and paraffin-embedded mouse liver tissue samples in the same manner as in Example 1, and used in the experiment. The detection gene is the same as in Example 1, using Cyp1a2 and FITC-labeled 4 kinds of single-stranded oligomeric DNA probes shown in sequence numbers 1 to 4 at both ends, and the respective concentrations of the 4 kinds of probes are 0 nM (nano mol), 1 nM, 2 nM, 3 nM, 4 nM, and 5 nM of various probe concentrations, the pseudo-oligo DNA (single-stranded) shown in SEQ ID NO: 5 was used at a concentration of 8 times the addition ratio. In addition, salmon sperm DNA from Invitrogen (catalogue number 15632-011, Salmon Sperm DNA solution) was added to a final concentration of 100 ug / ml (corre...

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Abstract

The invention provides an RNA in situ hybridization which is characterized by identifying the presence of target gene mRNA by hybridizing the target gene mRNA expressed in a tissue sample with at least one oligonucleic acid probe and detecting a low-molecular-weight compound label added to at least one nucleotide in the oligonucleic acid probe. In the method, the tissue sample is pretreated with at least one dummy oligonucleic acid (prehybridization), and subsequently the oligonucleic acid probe is contacted with the tissue sample to hybridize the oligonucleic acid probe with the target gene mRNA, or alternatively, a mixed solution of the oligonucleic acid probe and the dummy oligonucleic acid is contacted with the tissue sample to hybridize the oligonucleic acid probe with the target gene mRNA. The dummy oligonucleic acid has almost the same length as that of the oligonucleic acid probe. The dummy oligonucleic acid cannot hybridize with a region on the target gene mRNA with which the oligonucleic acid probe can hybridize, and cannot hybridize with the oligonucleic acid probe.

Description

technical field [0001] The present invention relates to RNA in situ hybridization for simple detection and quantification of gene expression in pathology and histochemistry, which can be used for research and diagnosis. Background technique [0002] (1) RNA in situ hybridization [0003] Various methods for pathological and histochemical detection of gene expression are known. Among them, there is in situ hybridization as a method for detecting the presence of mRNA in real time (in situ) on messenger RNA (mRNA), which is a transcription product formed by gene transcription. In this method, a buffer for in situ hybridization (Patent Document 1) in which a nucleic acid probe having a nucleic acid sequence complementary to the mRNA sequence to be detected is dissolved (Patent Document 1) is added to a tissue sample, and the nucleic acid probe and mRNA hybridization. In this case, a detection label is added to the nucleic acid probe, the label is detected by an appropriate me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09G01N33/58
CPCC12Q1/6832C12Q1/6841C12Q2543/10C12Q2527/125C12Q2545/101
Inventor 土居洋文松冈雅裕中野龙心永吉千鹤
Owner CELISH FD
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