Leica BIOSYSTEMS BOND Ready-to-Use ISH RNA Negative Control Probe PB0809 Mode D'emploi page 4

Instructions for Use
RNA Negative Control Probe was developed for use on the automated BOND system (includes Leica BOND-MAX system and Leica
BOND-III system) in combination with Anti-Fluorescein Antibody, BOND Polymer Refine Detection and BOND ancillary reagents. The
recommended staining protocol for RNA Negative Control Probe is ISH Protocol A. Enzyme retrieval is recommended using the BOND
Enzyme Pre-treatment Kit, Enzyme 1, for 15 minutes.
Appropriate tissue and reagent controls should always be used. The protocol for the tissue and reagent controls should correspond to
that of the RNA test probe.
Quality Control
Differences in tissue processing and technical procedures in the user's laboratory may produce significant variability in results,
necessitating regular performance of in-house controls in addition to the following procedures.
Positive Tissue Control
Used to indicate correctly prepared tissues and proper staining techniques. One positive tissue control should be included for each set of
test conditions in each staining run.
A tissue with weak positive staining is more suitable than a tissue with strong positive staining for optimal quality control and to detect
minor levels of reagent degradation.
Negative Tissue Control
Should be examined after the positive tissue control to verify the specificity of the labeling of probe to the target. Alternatively, the variety
of different cell types present in most tissue sections frequently offers negative control sites, but this should be verified by the user.
Negative Reagent Control
Use RNA Negative Control Probe PB0809 in place of the RNA test probe with a section of each patient specimen to evaluate
non-specific staining and allow better interpretation of specific staining at the target.
Positive Reagent Control
Use RNA Positive Control Probe PB0785 in place of the RNA test probe with a section of each patient specimen to provide information
on the preservation of nucleic acids in the tissue as well as accessibility of nucleic acids to the probe. If the RNA Positive Control Probe
fails to demonstrate positive staining, results with the test specimens should be considered invalid.
Patient Tissue
Examine patient specimens stained with RNA test probe last. Positive staining intensity should be assessed within the context of any
non-specific background staining of the RNA Negative Control Probe PB0809.
Results Expected
Normal Tissues
Using PB0809 no staining was observed in a wide range of tissues. (Total number of normal cases evaluated = 96).
Abnormal Tissues
Using PB0809 no staining was observed in ovarian tumors (0/3), thyroid tumors (0/3), lung tumors (0/3), liver tumors (0/3), brain tumors
(0/2), esophageal tumors (0/2), breast tumors (0/2), skin tumors (0/2), metastatic tumors of unknown origin (0/2), stomach tumors (0/1),
colon tumors (0/1), rectal tumors (0/1), a tumor of the larynx (0/1), and a tumor of the thymus (0/1) (Total number of abnormal cases
evaluated = 29).
PB0809 is recommended to aid as a screening tool to evaluate staining of an RNA test probe on a patient specimen.
Product Specific Limitations
RNA Negative Control Probe has been optimized at Leica Biosystems for use with Anti-Fluorescein antibody, BOND Polymer Refine
Detection and BOND ancillary reagents. Users who deviate from recommended test procedures must accept responsibility for
interpretation of patient results under these circumstances. The protocol times may vary, due to variation in tissue fixation and the
effectiveness of enzymatic digestion, and must be determined empirically. RNA Negative Control Probe should be used when optimising
retrieval conditions and protocol times.
Troubleshooting
Reference 3 may aid in remedial action.
Contact your local distributor or the regional office of Leica Biosystems to report unusual staining.
Further Information
Further information on in situ hybridization with BOND reagents, under the headings Principle of the Procedure, Materials Required,
Specimen Preparation, Quality Control, Assay Verification, Interpretation of Staining, Key to Symbols on Labels, and General Limitations
can be found in "Using BOND Reagents" in your BOND user documentation.
References
1. Pringle JH, Primrose L, Kind CN, et al. In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed
paraffin sections using a biotinylated oligonucleotide poly d(T) probe. Journal of Pathology. 1989;158(4):279—286.
2. Lewin B. Units of transcription and translation: the relationship between heterogeneous nuclear RNA and messenger RNA. Cell.
1975;4:11—20.
3. Clinical Laboratory Improvement Amendments of 1988, Final Rule 57 FR 7163 February 28, 1992.
4. Villanova PA. National Committee for Clinical Laboratory Standards (NCCLS). Protection of laboratory workers from infectious
diseases transmitted by blood and tissue; proposed guideline. 1991; 7(9). Order code M29-P.
5. Wilkinson DG. The theory and practice of in situ hybridization. In: Wilkinson DG. (ed.) In situ Hybridization A practical approach. 2
Edition. New York: Oxford University Press, 1998, pp.18—20.
Date of Issue
26 February 2020
PB0809
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