Detection method of small non-messenger RNA

A non-coding and qualitative detection technology, applied in the field of non-coding small RNA detection, can solve the problems of increasing the difficulty of low-abundance non-coding small RNA detection, increasing the risk of small RNA degradation, and restricting promotion and application. Ease of follow-up detection, reduction of experimental costs, and wide application

Inactive Publication Date: 2013-03-06
NO 3 PEOPLE HOSPITAL AFFILIATED TO SHANGHAI JIAOTOG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole process is cumbersome and time-consuming, and takes 2-3 days to complete; more importantly, isotopes are used in the detection process, which requires specific operating sites, equipment and related professionals. These shortcomings greatly limit its promotion and development. application
Therefore, some people use non-isotope-labeled probes (biotin-labeled probes) to replace isotope-labeled probes. Although the disadvantages of isotopes are avoided, the cumbersome operation process has not been reduced, and the time for detecting signals has not been shortened.
As we all know, RNases are ubiquitous, cumbersome operations increase the risk of small RNAs being degraded by RNases, which inevitably reduces the sensitivity of traditional Northern blot techniques for detecting small RNAs, especially increasing the detection of low-abundance non-coding small RNAs difficulty

Method used

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  • Detection method of small non-messenger RNA
  • Detection method of small non-messenger RNA
  • Detection method of small non-messenger RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] step 1

[0037] Synthetic oligonucleotide sequence: 5'-ATTTGCGTGTCATCCTTGCG-3', this sequence is completely complementary to the U6 sequence, the probe is short, and it is easy to bind and pair with the complementary sequence.

[0038] Using terminal transferase (TdT) to label biotin at the 3' end of the oligonucleotide, the reaction system is as follows:

[0039] Ultrapure water 25μl

[0040] 5-fold dilution TdT reaction buffer 10μl

[0041] 5μl of oligonucleotide probe to be labeled

[0042] Biotin-11-UTP 5μl

[0043] TdT (2U / μl) 5μl

[0044] Reaction process: water bath at 37°C for 30min, then add 2.5μl EDTA (0.2M) to stop the reaction, add 50μl chloroform:isoamyl alcohol (24:1) mixed reagent, mix well and centrifuge (12000g, 2-3min), take the supernatant 40-50μl for storage.

[0045] After the probe labeling is completed, the labeling effectiveness is tested: take different concentrations of labeled probe 1 μM, 2 μM, 5 μM, and 10 μM, add the loading buffer, ...

Embodiment 2

[0049] Referring to Example 1, different annealing conditions are used for liquid phase hybridization in this example.

[0050] Annealing condition 1: Set the temperature gradually on the PCR machine, 95 ° C for 2 min, and then drop 1 ° C every 90s until the temperature is 25 ° C, and maintain for a long time.

[0051] Annealing condition two: water bath at 95°C for 5min; then at 42°C for 2-3 hours.

[0052] refer to image 3 , the results show that the probe and target RNA can be hybridized to form a hybrid chain under two different liquid phase hybridization conditions. In terms of operation time, the action time of the PCR instrument is shorter than that of the water bath, the cooling process is mild, and the automatic control does not require human intervention; the action time of the water bath is prolonged, and the annealing is relatively mild. It is beautiful and clear, and non-specific binding is less than PCR instrument conditions. In short, both the water bath and...

Embodiment 3

[0054] Referring to Example 1, in this example, different hybridization buffers were used for liquid phase hybridization.

[0055] Hybridization buffer A: 30mmol / L phosphate buffer, 0.3mmol / L NaCl, 10mmol / LEDTA

[0056] Hybridization Buffer B: Tris-HCl (pH 8.5) 100mM, KCl 500nM, MgCl 2 15mM, 1% Tritox-100

[0057] Hybridization Buffer C: Tris-HCl (pH 7.5) 100 mM, 1 M NaCl, 10 mM EDTA

[0058] Hybridization buffer D: DEPC-treated H 2 O

[0059] Take 5 μg of fresh total RNA samples, 5 μl of U6 probe 4 groups, add equal volumes of different hybridization buffers, and place in a water bath at 95°C for 5 minutes; then at 42°C for 2-3 hours, fully hybridize, and then 15% non-denaturation Polyacrylamide gel electrophoresis detection.

[0060] refer to Figure 4 , the results show that under the same renaturation conditions, both phosphate buffer and Tris-HCl buffer can be used for liquid phase hybridization buffer, but the hybridization effect of phosphate buffer is obviously ...

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Abstract

The invention provides a detection method of small non-messenger RNA. The method includes: conducting non-isotope labelling at the 3' end of a oligonucleotide sequence probe under the effect of a terminal transferase; reacting the labelled probe and a sample RNA fully in a liquid phase hybridization environment to make the target small non-messenger RNA in the sample and the probe form heteroduplex; then subjecting the hybridization product to non-denaturing polyacrylamide gel electrophoresis, separating RNA-probes and non-hybridized excessive probes combined into double chains, transferring the heteroduplex to a nylon membrane, performing ultraviolet crosslinking fixation, adding streptavidin-HRP, thus achieving color developing and luminescence; and in the system, with the hybridized target RNA-probe and a single non-hybridized probe both being able to pass the streptavidin-HRP, adding an enzyme substrate to emit a fluorescence band, and performing developing on an X-ray plate.

Description

technical field [0001] The invention relates to a method for detecting non-coding small RNA, in particular to a method for detecting non-coding small RNA by using non-isotope labeling probe liquid phase hybridization. Background technique [0002] Non-coding small RAN (small non-messenger RNA, also referred to as small RNA in the present invention) is a large class of RNA composed of dozens of nucleotides to hundreds of nucleotides in cells that does not encode proteins, such as nuclear small RNA , nucleolar RNA, micro RNA, interfering small RNA, timing small RNA, etc. This type of RNA itself or combined with protein to form a complex has important biological functions. Studies have found that small RNA plays an important role in the development, It plays a vital role in growth, differentiation, and even disease occurrence, as well as virus invasion and defense. [0003] At present, the technologies that can be used to detect small RNA mainly include Northern blot, RT-PCR a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N27/447
Inventor 高丰厚郭跃辉姜斌
Owner NO 3 PEOPLE HOSPITAL AFFILIATED TO SHANGHAI JIAOTOG UNIV SCHOOL OF MEDICINE
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