SYSMEX ogt CytoCell RU-LPA 005 Instructions For Use Manual

Instructions For Use
REF: RU-LPA 005
Prenatal 13, 18 and 21 Enumeration Probe kit
Research Use Only
Further information available at www.ogt.com
Fluorescence In Situ Hybridisation (FISH) is a technique that allows DNA
sequences to be detected on metaphase chromosomes or in interphase nuclei
from fixed cytogenetic samples. The technique uses DNA probes that hybridise to
entire chromosomes or single unique sequences, and serves as a powerful
adjunct to classic cytogenetics. Recent developments have meant that this
valuable technique can now be applied as an essential tool in prenatal,
haematological and pathological chromosomal analysis. Target DNA, after
fixation and denaturation, is available for annealing to a similarly denatured,
fluorescently labelled DNA probe, which has a complementary sequence.
Following hybridisation, unbound and non-specifically bound DNA probe is
removed and the DNA is counterstained for visualisation. Fluorescence
microscopy then allows the visualisation of the hybridised probe on the target
material.
Intended Use
This product is intended to be used for research use only and is not for use in
diagnostic procedures.
Probe Specification
13 unique sequence, 13q14.2 Green
18 centromere, 18p11.1 – q11.1 (D18Z1) Blue
21 unique sequence, 21q22.13 Orange
The probe set 13, 18 and 21 is a mixture of green, blue and orange directly
labelled fluorescent DNA probes. The 18 centromere probe is specific for the
alpha satellite DNA sequences at the D18Z1 region of the chromosome 18. The
chromosome 13 probe includes RB1 gene as well as markers D13S1195,
D13S1155 and D13S915. The chromosome 21 probe includes markers D21S270,
D21S1867, D21S337, D21S1425, D21S1444 and D21S341.
Materials Provided
Probe: 50µl per vial or 100µl per vial
The probe is provided premixed in hybridisation solution (Formamide; Dextran
Sulphate; SSC) and is ready to use.
Counterstain: 150µl per vial
The counterstain is DAPI antifade (ES: 0.125µg/ml DAPI (4,6-diamidino-2-
phenylindole)).
Warnings and Precautions
1. For research use only. Not for use in diagnostic procedures. For professional
use only.
2. Wear gloves when handling DNA probes and DAPI counterstain.
3. Probe mixtures contain formamide, which is a teratogen; do not breathe
fumes or allow skin contact. Wear gloves, a lab coat, and handle in a fume
hood. Upon disposal, flush with a large volume of water.
4. DAPI is a potential carcinogen. Handle with care; wear gloves and a lab coat.
Upon disposal, flush with a large volume of water.
5. All hazardous materials should be disposed of according to your institution's
guidelines for hazardous waste disposal.
Storage and Handling
The kit should be stored between -25ºC to -15ºC in a freezer until the expiry date
indicated on the kit label. The probe and counterstain vials must be stored in the
dark.
Protocol Recommendations
Equipment Necessary but not Supplied
1. Hotplate (with a solid plate and accurate temperature control up to 80ºC).
2. Variable volume micropipettes and tips range 1µl - 200µl.
3. Water bath with accurate temperature control at 37ºC and 72ºC.
4. Microcentrifuge tubes (0.5ml).
5. Fluorescence microscope
Recommendation section).
6. Plastic or glass coplin jars.
7. Forceps.
8. Fluorescence grade microscope lens immersion oil.
9. Bench top centrifuge.
10. Microscope slides.
11. 24x24mm coverslips.
12. Timer.
13. 37ºC incubator.
14. Rubber solution glue.
Fluorescence Microscope Recommendation
For optimal visualisation of the probe we recommend a 100 watt mercury lamp
and plan apochromat objectives x63 or x100. The blue fluorophore has specificity
to the Aqua and DEAC spectrum (single bandpass Aqua or DEAC filter is
required).
Sample Preparation
Sample preparation should be performed according to the laboratory or institution
guidelines.
Amniotic fluid samples that appear bloody or brown should not be used, since
they may contain maternal blood and may lead to false results.
Suggested Protocol
Preparation of fresh amniotic fluid samples for FISH:
1. Centrifuge 2 – 5ml of whole amniotic fluid specimen for 7 minutes at 180xg,
carefully remove the supernatant without disturbing the cell pellet.
2. Resuspend the pellet in 2ml of 0.075M KCl. Leave at room temperature (RT)
for 5 minutes.
3. Add 2ml of fresh fixative (3:1 methanol:glacial acetic acid) to the
cells/hypotonic solution, adding the first ml dropwise whilst continuously
mixing. Mix well.
4. Centrifuge the suspension for 5 minutes at 280xg, carefully remove the
supernatant and resuspend the pellet in 2ml of fresh fixative.
5. Fixed specimens can be stored at this stage in a freezer at -20ºC.
6. If the sample is not to be frozen, centrifuge the tube at 280xg for 5 minutes.
Remove as much supernatant as possible without disturbing the cell pellet.
Flick the tube to resuspend the pellet in the small amount of fluid remaining.
7. To prepare slides for FISH, spot the cell suspension directly onto slide. Allow
to air dry.
Recommended slide Pretreatment:
1. Immerse the slide prepared from uncultured amniocytes in 2xSSC for 1 hour]
at 37ºC.
2. Place the slide in freshly made pepsin working solution (5mg of pepsin added
to 100ml of 0.01M HCl) for 13 minutes at 37ºC.
3. Immerse the slide in phosphate buffered saline (PBS) at RT for 5 minutes.
4. Immerse the slide in post fixation solution (0.95% formaldehyde: 1.0ml of
37% formaldehyde, 0.18g of MgCl
5. Immerse the slide in PBS at RT for 5 minutes.
6. Immerse the slide in 70% ethanol at RT. Allow the slide to stand in the
ethanol wash for 1 minute.
7. Remove the slide from 70% ethanol. Repeat step 6 with 85% ethanol,
followed by 100% ethanol.
8. Allow to air dry.
FISH Protocol
(Note: Please ensure that exposure of the probe to laboratory lights is limited at
all times)
DS144/RUO V005.00/2021-05-05 (A001 v3 / A002 v2 / A005 v3)
(Please see Fluorescence Microscope
and 39.0ml of PBS) for 5 minutes at RT.
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