RNA in situ hybridization experimental steps - Database & Sql Blog Articles

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RNA in situ hybridization experimental procedure,

1. Transcription buffer: 200 mmol/I Tris-HCl (pH 7.5), 30 mmol/L MgCl

, 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/LUTP, 0.32 mmol/L fluorescein-11-UTP dissolved in sterile water.

7. RNase I without RNase: freshly prepared with sterile water and diluted to 10 U/ 80 μl.

8. 4 mol/L NaHCO

, autoclaved.

9. 6 mol/L NaHCO

, autoclaved.

10. Glacial acetic acid.

11. Autoclave with 3 mol/L sodium acetate (pH 5.2).

12. 10 mg/ml yeast tRNA.

13. Hybridization buffer: Standard hybridization buffer can be used. An optimized buffer solution is recommended here and is available from the Amersham clause. The buffer consisted of 4XSSC, 600 μg/ml salmon sperm DNA, 2X Denhardt solution. This hybridization buffer also contains a proprietary compound for increasing the efficiency of hybridization. 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), soluble in TBS.

18. Anti-fluorescein-alkaline phosphate enzyme conjugate: commercially available from Amersham Corporation.

19. Detection buffer: 100 mmol/L Tris-HCl (pH 9.5), 100 mmol/L NaCl, 10 mmol/L MgCl

.

20. NBT solution: 50 mg/ml Niroblue tetrazolium (NBT) was dissolved in dimethylformamide.

21. BCIP solution: 75 mg/ml bromo-chloro-indolyl phosphate (BCIP) is dissolved in 70% (v/v) dimethylformamide.

RNA in situ hybridization experimental steps, second, method of operation

Probe mark

(1) Add the following reagents in a 1.5 ml microcentrifuge tube at room temperature: transcription buffer 4 μl, 0.2 mg/L DTT 1 μl, HPRI 20U, nucleotide solution 8 μl, linear DNA template 1 μg, RNA polymerization Enzyme 25 U, add water to a final volume of 20 μl and mix gently.

(2) Keep at least 2 h. Different RNA polymerases have different incubation temperatures, SP6 is 40 °C, and both T3 and T7 are 37 °C.

(3) Labeled RNA probes can be stored at -20 °C.

2. Purification and processing of labeled probes

(1) 10 U of RNase-free DNase I was added to the RNA probe mixture and incubated at 37 ° C for 10 min.

(2) Add 20 μl of 0.4 mol/L NaHCO

, 20 μl 0.6mol/L Na

CO

, 60 μl of sterilized water. Mix gently and heat at 60 ° C for alkaline hydrolysis. The incubation time depends on the length of the transcript and the size of the probe required.

(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 place at -20 °C for at least 2 h to precipitate RNA.

(4) Precipitate RNA by centrifugation at the highest speed for 15 min on a microcentrifuge and discard the supernatant.

(5) Rinse the pellet with 70% ethanol, keep at -20 °C: centrifuge for 5 min, discard the supernatant.

(6) Dissolve the RNA probe with sterile water and adjust the RNA probe concentration to 10-20 times the concentration required for hybridization, and store at -20 °C.

3. Tissue and cell pretreatment

All tissues used for in situ hybridization can be fixed with neutral buffered formaldehyde (4%). The aldehyde fixative can retain the cellular RNA well. The formaldehyde perfused animal or freshly frozen tissue sections should be fixed in formaldehyde at room temperature. The perfused tissue is easier to freeze and slice than the perfused tissue, making it easier to attach to the slide.

(1) The tissue that is perfused.

The flow was filled with normal saline to remove red blood cells, and then the animals were irrigated with about 200 ml of a 4% formaldehyde solution dissolved in 0.1 mol/L PBS and placed in a 20% sucrose (dissolved in PBS) solution overnight. The tissue was frozen in liquid nitrogen and stored at -80 °C.

Tissues were sectioned (10 μm or thinner) at approximately -20 ° C and sections were transferred to polylysine-treated slides. Tissue sections were stored at -80 °C.

(2) Unperfused tissue.

Fresh tissue was lyophilized in liquid nitrogen or lyophilized in isopentane cooled to -30 °C. Tissues were sectioned and transferred to polylysine treated slides. Tissue sections were stored at -80 °C.

Slides were removed from -80 °C prior to hybridization and placed directly in 4% formaldehyde for 1 h at room temperature. Wash thoroughly with PBS or 2X SSC.

RNA in situ hybridization experimental procedure,

For any ISH experiment, this step is most important for a successful outcome. Every hybrid system requires its own optimal pretreatment protocol. The design of the pretreatment is based on the principle that both the probe and the antibody conjugate can be easily contacted to the target molecule, the tissue can be maintained in its original morphological structure, and the target can be maintained in its original position. In addition, the pretreatment protocol can also be used to set up some of the control experiments necessary for ISH.

4. Hybridization and washing

Control of the hybridization string can be controlled by the temperature of the hybridization step, the salt concentration, and the concentration of the amide. However, more convenient control is to apply these parameters to the rigorous washing after hybridization.

(1) The hybridization buffer was preheated at 37 ° C for 15 min, diluted with deionized formamide in a ratio of 1:1, and thoroughly mixed.

(2) Add the required amount of probe preheated at 55 °C to the hybridization buffer. Probe concentrations from 300 to 600 ng/ml are suitable for most hybridizations.

(3) Cover the sections with an appropriate volume of hybridization buffer/probe mixture.

(4) Place the slice on the hot plate and place it for 3-6 minutes. The holding time depends on the type of target.

(5) Place the sections in a wet box and mix at the desired temperature (typically 55 ° C) and time.

(6) Wash the slides to the required degree of rigor. Typical washing procedures include: 1X SSC-0.1% (V/V) SDS wash twice at room temperature for 5 min each time; 0.2XSSC-0.1% (V/V) SDS Wash twice at 55 °C for 10 times each time Min.

(7) To reduce the background, RNA probes can be further processed with RNase. This step only removes unhybridized single-stranded probes. The specific steps are as follows: After the slide was rinsed in 2X SSC for 2 min, it was incubated at 37 ° C for 20 to 30 min in preheated 2X SSC solution (containing RNase A 10 μg/ml). The slides were rinsed in 2XSSC before proceeding to the next step.

5. Blocking, antibody incubation and washing

RNA in situ hybridization experimental procedure,

The slides should be gently shaken in the following series of insulations, unless otherwise stated, at room temperature. Note: The TBS used here has a higher concentration of salt than the standard TBS.

(1) The slide was washed in TBS for 5 min.

(2) The slides were incubated for 1 h in the blocking solution.

(3) Rinse the slides in TBS for 1 min, drain the buffer on the slides, and, if necessary, blot the liquid around the surface of each slide.

(4) The anti-fluorescein alkaline phosphatase conjugate was diluted with TBS containing 0.5% (m/V) BSA fraction V at a ratio of 1:10000. The sheets were covered with antibodies (100 μl per slide) and allowed to stand for 1 h.

(5) The slides were washed 3 times in TBS for 5 min each time.

6. Detection

(1) Wash the slides in the assay buffer for 5 min, drain the liquid from each slide, and, if necessary, blot the liquid around the surface of the slide.

(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 section and place it in the dark for 4 to 24 hours.

(3) Rinse the slides twice with distilled water for 2 min each time.

(4) Counterstain if necessary. Add the cover and cover the coverslip.

(5) Observe the results under an optical microscope.

RNA in situ hybridization experimental procedure