C11orf95/RELA

Product Name

C11ORF95/RELA gene fusion t(11;11) probe reagent.

Package Specifications

10 Tests/box

Intended use

The reagent carries out in situ hybridization staining on the basis of routine staining to provide doctors with auxiliary information for diagnosis. The test results are only for clinical reference and should not be used as the only basis for clinical diagnosis. Clinicians should comprehensively judge the test results in combination with the patient’s condition, drug indications, treatment response and other laboratory test indicators.

Detection principle

Fluorescence in situ hybridization is a technique for directly observing specific nucleic acids in cells in vitro. According to the principle of base complementary pairing, the specific probe is complementary to the target sequence in the cell. Due to the fluorescence of the probe,the gene state of the hybrid probe and the target sequence can be clearly observed under the fluorescence microscope under the appropriate excitation light.

Product Main Components

The kit consists of C11ORF95/RELA dual-color probe, as shown in Table 1.

Storage conditions & Validity

Keep sealed away from light at -20°C±5°C, and the validity period is 12 months.After the cover is opened, it can be sealed and stored in 2~8°C away from light within 24 hours. After the cover is opened, it should be sealed and stored in -20±5°C away from light for a long time. Transport with temperature below 0°C.

Applicable Instruments

Fluorescence microscopy imaging systems, including fluorescence microscopy and filter sets suitable for DAPI (367/452), Green (495/517), and Orange (547/565).

Sample Requirements

1. Applicable specimen types: Paraffin-embedded specimensfor surgical resection or biopsy.
2. Tissue should be fixed with 4% neutral formaldehyde fixation solution within 1 hour after in vitro, and the tissue should be fixed by conventional dehydration and paraffin embedding.

Test method

1. Pre-hybridization or Pretreatment
   1. Baking: Slides heating at 80°C for 30 min or 65°C for 2 hours or overnight.
   2. Dewaxing: According to the customer laboratory protocol (commonly with Xylene for 15 min).
   3. Hydration: Take out the slides and put them respectively into 100%, 85%, and 70% EtOH at room temperature for 3 minutes each. Immerse them in deionized water for 3 minutes and remove excess water by air-drying.
   4. Permeation: Immerse the slides in deionized water at 100°C and boil continuously for 20-40 minutes (conventional 20 min). Remove excess water by air-drying.
   5. Digestion: Protease enzymatic digestion at 37°C for 10-40 minutes. Mix the protease work buffer (50mmol HCl) and the 10x protease solution (Pepsin concentration 0.5%) in a proportion of 9:1 to prepare the enzymatic digestion solution.
   6. Washing: Wash with 2xSSC at room temperature for 5 minutes.
   7. Dehydration: Take out the slides and dehydrate in 70%, 85%, and 100% gradient ethanol at room temperature for 2 minutes each time. Remove excess EtOH solution by air-drying.
2. Denaturation and Hybridization (Performed in a darkroom)
   1. Take out the probe, leave it at room temperature for 5 min, turn it upside down with force, mix it well, and centrifuge it briefly (do not use a vortex instrument). Drop 10μL into the hybridization area, cover with a 22mm × 22mm cover glass immediately, ensuring no bubbles, and seal the edges with rubber glue.
   2. Place the slides in the hybridizer, denature at 85°C for 5 min (preheat the hybridizer to 85°C), and hybridize at 42°C for 2-16 hours.
3. Washing (Performed in a darkroom)
   1. Carefully remove the sealing glue around the cover glass with tweezers to avoid shifting. Immerse the sample in 2xSSC for about 5 seconds, then gently push a corner of the cover glass to the edge of the slide and remove it with tweezers.
   2. Place the sample in 2xSSC at room temperature for 1 min.
   3. Take out the slides and immerse in a preheated at 68°C 0.3% NP 40/0.4xSSC (Preparation of 0.3% NP-40/0.4xSSC: For 1L preparation,take 3mL NP 40 and 20mL 20xSSC, dissolve fully, mix well, and use 1M NaOH to adjust the pH to 7.2) solution and wash for 2min.
   4. Take out the sample and immerse it in deionized water preheated at 37oC in advance for 1min; dry it naturally in the dark place.
4. Counterstaining (Performed in a darkroom)
   The following operations should be performed in a darkroom
   10μl DAPI compound dye is dropped in the hybridization area of the glass slide and immediately covered. The suitable filter is selected for glass slide observation under the fluorescence microscope.
5. FISH Results Observation
   Place the counterstained film under the fluorescence microscope, and first put it under the low-power objective lens (10x) confirm the cell area under the microscope; Go to 40x under the objective lens, find a position where the cells are evenly distributed; Then in the high-power objective (100x) the FISH results of nuclei were observed.

Common Signal Type Interpretation

Limitations of test methods

1. The results of this kit may be affected by various factors, including the characteristics of the sample, hybridization temperature and time, operating environment, and the limitations of current molecular biology technology. These factors may lead to incorrect results.
2. Users must understand the potential errors and accuracy limitations that may exist in the detection process.

Precautions

1. This product is for in vitro diagnosis only.
2. Please read this manual carefully before testing. The testing personnel must receive professional technical training, and signal counting personnel must be able to observe and distinguish orange and green signals.
3. When testing clinical samples, if hybridization signal counting is difficult, the sample is insufficient for a repeat test, or the cell volume is inadequate for analysis, the test will not provide results.
4. The DAPI counterstaining agent used in this experiment has potential toxicity or carcinogenicity. It must be handled in a fume hood, and personnel should wear masks and gloves to avoid direct contact.
5. All chemicals are potentially dangerous. Avoid direct contact. Used kits are clinical waste and should be properly disposed of.

Manual Approval date & Revision date

V1. 0: Approval date: October 24, 2019.
V1. 2: Revision date: December 7, 2021.