RNA in situ hybridization assay
In situ hybridization (ISH), also known as hybrid histochemistry or hybridization for cytology, is a technique that enables morphological demonstration of the presence of specific DNA or RNA sequences in prepared individual cells, tissue fractions, single cells, or chromosomes. In situ hybridization is the only method for studying the cellular localization of DNA and RNA sequences in heterogeneous cell populations. This experiment is derived from the "RNA Laboratory Guidebook" edited by Xiaofei Zheng.
Operation method
5.7 RNA in situ hybridization
Principle
In situ hybridization is based on the principle that labeled single-stranded DNA or RNA fragments containing complementary sequences (probes) hybridize with cellular DNA or RNA under appropriate conditions to form stable hybrids. Both DNA (dsDNA, oligonucleotide, and ssDNA) and RNA (SSC RNA) probes can be used to localize DNA and mRNA, and both can be used in two main types of labeling strategies: direct labeling, in which a reporter molecule (e.g., an isotope) is attached to the DNA or RNA; and indirect labeling, in which a semi-antigen (e.g., biotin or digoxin) is attached to the probe, followed by a labeled conjugate protein (e.g., a protein of the same type). The target probe hybrid is then detected with a labeled binding protein (e.g., affinity protein) or with an antibody specific to the target probe. These basic steps are interdependent, and changes in any of the previous steps require corresponding changes in the subsequent steps in order to obtain optimal results. In fact, for many histochemistry steps, the "ideal method" for a given system must be established by extensive practice.
Materials and Instruments
Linear DNA template DNaseⅠ Antifluorescein-alkaline phosphatase conjugate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Transcription Buffer Human Placental Ribonuclease Inhibitor DTT Nucleotide Solution Ice Acetic Acid NaHCO3 Sodium Acetate Yeast tRNA Hybridization Buffer SSC Storage Buffer Closure Buffer Assay Buffer NBT Solution BCIP Solution
1. Transcription buffer: 200 mmol/I Tris-HCl ( pH 7.5), 30 mmol/L MgCl2, 10 mmol/L spermidine, 0.5% (m/V) BSA.
2. human placental ribonuclease inhibitor (HPRT) : 20 U/μl.
3. 2 mmol/L DTT: freshly prepared, filtered and sterilized.
4. linear DNA template: usually 0.5~1 μg/μl.
5. SP6, T7 or T3 RNA polymerase.
6. nucleotide solution: 1.25 mmol/L ATP, 1.25 mmol/L GTP, 1.25 mmol/L CTP, 0.312 mmol/L UTP, 0.312 mmol/L fluorescein-11-UTP in sterilized water.
7. DNase I without RNase: freshly prepared in sterilized water and diluted to 10 U/ 80 μl.
8. 4 mol/L NaHCO3, autoclaved.
9. 6 mol/L NaHCO3, autoclave.
10. glacial acetic acid.
11. 3 mol/L sodium acetate (pH 5.2) autoclaved.
12. 10 mg/ml yeast tRNA.
13. Hybridization buffer: Standard hybridization buffers are available. A finished optimized buffer, available from Amersham, is recommended. The buffer consists of 4XSSC, 600 μg/ml salmon sperm DNA, and 2X Denhardt's solution. This hybridization buffer also contains a proprietary compound to improve hybridization efficiency. Prior to use, this buffer should be diluted 1:1 with deionized formamide and the diluted solution can be stored at -20°C.
14. 20X SSC stock solution: 3 mol/L NaCl, 0.3 mol/L sodium citrate (pH 7.0).
15. 10% (m/V) SDS.
16. TBS: 100 mmol/L Tris-HCl (pH 7.5 ), 400 mmoI/L NaCl.
17. blocking buffer: 0.5% (m/V) blocking agent (RPN3023, Amersham International), dissolved in TBS.
18. Antifluorescein-alkaline phosphatase conjugate: available from Amersham.
19. assay buffer: 100 mmol/L Tris-HCl (pH 9.5), 100 mmol/L NaCl, 10 mmol/L MgCl2.
20. NBT solution: 50 mg/ml of niroblue tetrazolium (NBT) was dissolved in dimethylformamide.
21. BCIP solution: 75 mg/ml bromo-chloro-indolyl phosphate (BCIP) dissolved in 70% (V/V) dimethylformamide.
II. Methods of operation
1. Probe labeling
(1) Add the following reagents into a 1.5 ml microcentrifuge tube at room temperature: 4 μl of transcription buffer, 1 μl of 0.2 mg/L DTT, 20 U of HPRI, 8 μl of nucleotide solution, 1 μg of linear DNA template, and 25 U of RNA polymerase, and then add water to the final volume of 20 μl, and then mix gently.
(2) Hold for at least 2 h. Different RNA polymerases have different holding temperatures, 40℃ for SP6, 37℃ for T3 and T7.
(3) The labeled RNA probe can be stored at -20℃.
2. Purification and processing of labeled probes
(1) Add 10 U of RNase-free DNase Ⅰ to the RNA probe mixture and hold at 37℃ for 10 min.
(2) Add 20 μl of 0.4 mol/L NaHCO3, 20 μl of 0.6 mol/L Na2CO3 and 60 μl of sterilized water. Mix gently and hold at 60 ℃ for alkaline hydrolysis. The holding time depends on the length of the transcript and the size of the desired probe.
(3) Add 1.3 μl glacial acetic acid, 20 μl 3 mol/L sodium acetate (pH 5.2), 2 μl 10 mg/ml yeast tRNA, 500 μl ethanol, and leave it at -20 ℃ for at least 2 h to precipitate RNA.
(4) Centrifuge the RNA in a microcentrifuge at maximum speed for 15 min and discard the supernatant.
(5) Wash the precipitate with 70% ethanol, keep at -20℃: centrifuge for 5 min, discard the supernatant.
(6) Dissolve the RNA probe in sterilized water and adjust the concentration of RNA probe to 10~20 times of the required hybridization concentration and store at -20℃.
3. Tissue and cell pretreatment
All tissues used for in situ hybridization can be fixed in neutral buffered formaldehyde (4%), the aldehyde fixative retains cellular RNA well, formaldehyde perfused animals or freshly frozen tissue sections should be fixed in formaldehyde at room temperature, unperfused tissues are easier to be frozen sectioned than perfused tissues, and easier to be adhered to slides.
(1) Perfused tissue.
Animals were perfused with normal saline to remove hemoglobinocytes, followed by irrigation with approximately 200 ml of 4% formaldehyde solution dissolved in 0.1 mol/L PBS and placed in 20% sucrose (dissolved in PBS) solution overnight. The tissues were frozen in liquid nitrogen and stored at -80°C.
Tissue is sectioned (10 μm or thinner) at approximately -20°C and the sections are transferred to polylysine-treated slides. Store tissue sections at -80°C.
(2) Unperfused tissue.
Lyophilize fresh tissue in liquid nitrogen or lyophilized and stored in isopentane cooled to -30°C. The tissue is sectioned and transferred to polylysine-treated slides. Store tissue sections at -80°C.
Remove slides from -80°C and place directly in 4% formaldehyde for 1 h at room temperature before hybridization. wash thoroughly with PBS or 2X SSC.
For any ISH experiment, this step is the most important for successful results. Each hybridization system requires its own optimal pretreatment protocol. The design of the pretreatment is based on the principle that the probe-antibody conjugate can easily reach the target molecule, while allowing the tissue to retain its original morphology and structure, and the target to remain in its original position. In addition, the pretreatment protocol can be used to set up control experiments necessary for ISH.
4. Hybridization and washing
Hybridization stringency can be controlled by the temperature, salt concentration and flat amide concentration of the plants in the hybridization step. However, a more convenient control is to apply these parameters to the post hybridization stringency wash.
(1) Preheat the hybridization buffer at 37℃ for 15 min, dilute with deionized formamide at 1:1 ratio and mix well.
(2) Add the desired amount of probe preheated at 55°C to the hybridization buffer. 300-600 ng/ml probe concentration is suitable for most hybridizations.
(3) Cover the section with the appropriate volume of hybridization buffer/probe mixture.
(4) Place the sections on a hot plate for 3-6 min, depending on the type of target.
(5) Place sections in a wet box and hybridize at the desired temperature (typically 55°C) and time.
(6) Wash slides to the desired degree of stringency. Typical washing procedures include: 1X SSC-0.1%(V/V) SDS twice at room temperature for 5 min each; 0.2X SSC-0.1%(V/V) SDS twice at 55°C for 10 min each.
(7) To reduce background, the RNA probe can be further processed with RNase. This step only removes the unhybridized single-stranded probes. The procedure is as follows: the slides are washed in 2X SSC for 2 min, then held in preheated 2X SSC (containing 10 μg/ml of RNase A) at 37°C for 20~30 min. Before proceeding to the next step, the slides are washed in 2X SSC.
5. Blocking, antibody holding and washing
The slides are gently shaken during the following series of incubations, which are performed at room temperature unless otherwise stated. Note: The TBS used here contains a higher salt concentration than standard TBS.
(1) Slides are washed in TBS for 5 min.
(2) Place the slide in a closed solution and hold for 1 h.
(3) Rinse the slides in TBS for 1 min, drain the buffer from the slides, and, if necessary, blot the liquid from the surface of each slide around the sections.
(4) Dilute the anti-luciferin alkaline phosphatase conjugate with TBS containing 0.5% (m/V) BSA component V at a ratio of 1:10,000. Cover the sections with the antibody (usually 100 μl per slide) and leave to incubate for 1 h. The antibody should be applied to the sections at a rate of 1:10,000.
(5) The slides were washed 3 times in TBS for 5 min each time.
6. Detection
(1) The slides are washed in assay buffer for 5 min, and the liquid is drained from each slide and, if necessary, pipetted around the sections on the slide surface.
(2) Add 45 μl of NBT stock solution and 35 μl of BCIP stock solution to 10 ml of assay buffer. Add 500 μl of detection reagent to each slide, and place it in the dark for 4~24 h for color development.
(3) Wash the slides twice with distilled water for 2 min each time.
(4) Re-stain if necessary. Add sealer and cover the coverslip.
(5) Observe the results under a light microscope.