Ultrahigh sensitivity RNA in-situ detection method

Abstract

The invention discloses an ultrahigh sensitivity RNA in-situ detection method. The method comprises the following steps: S1, obtaining a tissue slice through a frozen section; s2, fixing is conducted for 15 min through 4% PFA; s3, 2x SSC rinsing is carried out twice, and each rinsing is carried out for 5 min; s4, rinsing with 70% alcohol; s5, soaking in 70% alcohol for 2 hours; s6, carrying out # imgabs0 # treatment for 15 minutes; s7, PBST rinsing is carried out once; s8, protease K treatment is performed for 10 min; s9, rinsing twice with 4% BSA (Bovine Serum Albumin); s10, 2x SSC rinsing is carried out twice; s11, sealing is conducted for 30 min through sealing liquid; s12, performing probe hybridization for 2 hours; s13, cleaning with a cleaning solution twice, 0.5 h each time; s14, incubation is carried out for 0.5 h through stepdavidin-hrp (stem davidin-human recombinant protein); s15, 4% BSA cleaning is carried out five times, and each time is 5 min; s16, carrying out a TSA reaction for 15 min; s17, sheet sealing is carried out; and S18, observing and recording the signal through a microscope. The probe is divided into the primary probe and the secondary probe, the primary probe and the secondary probe are mixed to form the complete probe, a plurality of probes are designed for each RNA molecule, signal amplification is carried out, and signal amplification is carried out through combination of streptavidin coupled with HRP and biotin of the secondary probe and through a Tyramide signal amplification technology, so that signal detection with ultrahigh sensitivity can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device drying devices, and in particular to an ultra-high sensitivity RNA in-situ detection method. Background Art

[0002] RNA is a ubiquitous biomolecule in cells. It is transcribed and synthesized using DNA as a template, further serving as a template molecule to guide protein translation. Therefore, RNA plays a crucial role in converting genetic information into specific functional molecules. In modern molecular biology, the method of visualizing the spatiotemporal expression patterns of RNA molecules in different tissues and cells is called in situ hybridization. The development of in situ hybridization technology has played a crucial role in understanding gene regulation and function, as well as their mechanisms in development, regeneration, and disease.

[0003] The basic principle of RNA FISH is to use complementary labeled probes to hybridize with target RNA sequences to form stable probe-RNA hybrids, and then further indicate the location of the target RNA in tissues or cells by detecting the signal of the probe.

[0004] Classic RNA in situ hybridization mainly includes the following steps: (1) Synthesize the antisense DNA template strand of the target RNA; (2) Using the DNA as a template, RNA complementary to the target RNA sequence is synthesized by in vitro transcription, and digoxigenin or other markers are incorporated during the synthesis process; (3) Detection of digoxin by digoxin antibodies, which are usually conjugated to catalase or alkaline phosphatase; (4) Enzymatic reaction for further signal amplification; (5) Signal detection.

[0005] This method is sensitive to detection, but the experimental cycle is long, and it cannot achieve the sensitivity of detecting single-molecule RNA. The single-molecule RNA in situ hybridization technology developed later mainly improved the design and preparation methods of the probe. The core change is to replace the single long-chain probe with a short-chain probe, so that one RNA molecule can bind to multiple RNA probes. This short-chain probe is directly synthesized by chemical synthesis, and fluorescent molecules are directly added to both ends, thus eliminating the probe binding and subsequent signal detection steps, greatly shortening the experimental cycle. At the same time, it also increases the resolution of the signal, so that the resolution of the single molecule can be achieved. However, the disadvantage is that the signal intensity is sacrificed. When exploring the expression location of unknown RNA, especially when looking for expression in specific cells from tissues, this low fluorescence intensity and low signal greatly hinder the rapid detection of the signal. In addition, since each probe needs to be fluorescently labeled at both ends, its detection cost is much higher than that of traditional in situ hybridization technology.

[0006] To address the above issues, we propose an ultra-high sensitivity RNA in situ detection method. Summary of the Invention

[0007] The purpose of the present invention is to provide an ultra-high sensitivity RNA in situ detection method to solve the existing problems.

[0008] To achieve the above objectives, the present invention provides the following technical solution: an ultra-high sensitivity RNA in situ detection method, comprising the following steps: S1, obtain tissue sections by frozen section; S2, 4% PFA fixation for 15 min; S3, 2x SSC rinse twice, 5 min each time; S4, 70% alcohol rinse; S5. Soak in 70% alcohol for 2 hours; S6, Process for 15 minutes; S7, rinse once with PBST; S8, proteinase K treatment for 10 min; S9, 4% BSA rinse twice; Rinse twice with S10 and 2x SSC; S11, blocking solution for 30 min; S12, probe hybridization for 2 h; S13, clean twice with cleaning solution, each time for 0.5h; S14, stepdavidin-hrp incubation for 0.5 h; S15, 4% BSA wash 5 times, 5 min each time; S16, TSA reaction for 15 min; S17, sealing; S18. Observe and record the signal under a microscope.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention divides the probe into a primary probe and a secondary probe, mixes the two to form a complete probe, designs multiple probes for each RNA molecule, performs signal amplification, and combines streptavidin coupled with HRP with biotin in the secondary probe, and amplifies the signal through tyramide signal amplification technology, thereby achieving ultra-high sensitivity signal detection. The present invention absorbs the advantages of traditional in situ hybridization and single-molecule in situ hybridization technology, and circumvents the long experimental cycle and the problem of not being able to achieve the sensitivity of detecting single-molecule RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the probe segmentation process of the present invention; Figure 2 Schematic diagram of the cells of the present invention. DETAILED DESCRIPTION

[0011] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0012] Specific implementation case 1: An ultra-high sensitivity RNA in situ detection method specifically comprises the following steps: 1) The probe is divided into a primary probe and a secondary probe. The primary probe contains a gene-specific sequence and a sequence that is complementary to the secondary probe. The secondary probe contains a biotin modification for subsequent signal amplification. The two are mixed to form a complete probe ( Figure 1 A) 2) Design multiple probes for each RNA molecule to amplify the signal ( Figure 1 B); 3) By coupling HRP-conjugated streptavidin to the biotin of the secondary probe, and further amplifying the signal through tyramidesignal amplification technology, ultra-high sensitivity signal detection can be achieved.

[0013] Specific implementation case 2: An ultra-high sensitivity RNA in situ detection method comprises the following steps: S1, obtain tissue sections by frozen section; S2, 4% PFA fixation for 15 min; S3, 2x SSC rinse twice, 5 min each time; S4, 70% alcohol rinse; S5. Soak in 70% alcohol for 2 hours; S6, Process for 15 minutes; S7, rinse once with PBST; S8, proteinase K treatment for 10 min; S9, 4% BSA rinse twice; Rinse twice with S10 and 2x SSC; S11, blocking solution for 30 min; S12, probe hybridization for 2 h; S13, clean twice with cleaning solution, each time for 0.5h; S14, stepdavidin-hrp incubation for 0.5 h; S15, 4% BSA wash 5 times, 5 min each time; S16, TSA reaction for 15 min; S17, sealing; S18. Observe and record the signal under a microscope.

[0014] result: Combined with implementation cases 1 and 2, and Figure 2 As shown, the expression of scaffold35.g35 RNA in nematoblast cells of the non-model organism Hydrozoa viridis. DAPI is a nuclear stain, Scaffold35.g35 is the signal of the gene in nematoblast cells detected by this method, and Algae is the autofluorescence of symbiotic algae, marking endoderm cells.

[0015] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An ultra-high sensitivity RNA in situ detection method, characterized in that: The following steps are involved: S1, obtain tissue sections by frozen section; S2, 4% PFA fixation for 15 min; S3, 2x SSC rinse twice, 5 min each time; S4, 70% alcohol rinse; S5. Soak in 70% alcohol for 2 hours; S6, Process for 15 minutes; S7, rinse once with PBST; S8, proteinase K treatment for 10 min; S9, 4% BSA rinse twice; Rinse twice with S10 and 2x SSC; S11, blocking solution for 30 min; S12, probe hybridization for 2 h; S13, clean twice with cleaning solution, each time for 0.5h; S14, stepdavidin-hrp incubation for 0.5 h; S15, 4% BSA wash 5 times, 5 min each time; S16, TSA reaction for 15 min; S17, sealing; S18. Observe and record the signal under a microscope.

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