Tissue Microarrays In Situ Hybridization Flashcards
What are Tissue Microarrays?
This technique address the issues of
cumbersome nature of procedures, why
might this be useful?
Definition: Paraffin blocks in which up to 1000
separate tissue cores are assembled in
array fashion to allow multiplex histological analysis
This technique address the issues of
cumbersome nature of procedures, this might be useful because…
Tissue Microarrays Procedures include: (3 steps)
1.) Hollow needle removes tissue cores as
small as 0.6mm (areas of interest)
2.) Cores are inserted into a recipient block in
a precisely spaced pattern
3.) Sections cut normally with microtome
Tissue Microarrays Uses:
◦ Validation of a new technique on human tissue
- Very difficult process
- Often times there is not enough human sample to help validate the stain or technique
◦ Difficulty in obtaining tissue:
- Pathologist’s goal is to provide diagnostic consultation, not compile material for future experimentation
- Stringent guidelines for obtaining informed consent for its use
Tissue Microarrays Advantages:
1.) Amplification of a scarce resource
2.) Experimental uniformity (same conditions)
3.) Decreased assay volume
4.) Does not destroy original block for diagnosis
5.) Reduces reagents and time, sometimes to 1/60th
!!!!!! Valuable clinical and research resource !!!!!!
Tissue Microarrays Advantages:
6.) Average standard tissue block can yield appx 100 sections thus providing 100 assays
7.) TMA block can be punched on average 600 times, together with appx 100 sections thus providing up to 60,000 assays
8.) Each tissue sample is treated in an identical manner
9.) Very small amount of reagent used to analyze an entire cohort
What are some disadvantages of Tissue Microarrays?
1.) It is questioned if small spots are representative of a whole section?
- Skeptics claim amount analyzed is too small and potentially not representative of the entire tumor
- Data analysis shows that any given spot may be negative, the analysis of hundreds or thousands of cases eliminates the affect of variability of a single data point
- Yale University School of Medicine study on standard breast cancer prognostic markers (ER / PR / HER2) showed analysis of only 2 spots resulted in >95% accuracy
Tissue Microarrays: IHC with standard Formalin
Small tissue sections tend to oxidize over
time (as little as a week)
This destroys tissue antigenicity
Possible remedy includes not using a
water bath, instead tape method
Tissue Microarrays: Analysis
Results can be analyzed with advanced software
Data and images can be organized and stored for future reference
Can assist in collaboration between pathologists and research institutions
Tissue Microarrays: Examples of scanners
1.) Nanozoomer RS
2.) BLISS
3.) Nanozoomer HT
What is Polymerase Chain Reaction used for?
What can PCR be used to detect?
What is PCR done through?
Used to study specific targeted DNA
sequences through amplification
Can be used to detect bacteria, viruses, and
other markers useful in diagnosing cancers
and diseases
Is done through cycling temperatures with
specific steps to amplify DNA
What is needed for a Polymerase Chain Reaction?
1.) Specimen with potential gene sequence
2.) Nucleotides (A,T,G,C)
3.) Primers, to detect the target sequence
4.) Polymerase to produce a new strand of DNA
What is the first step in Polymerase Chain Reaction?
How does denaturation happen?
What temperature is DNA denaturation done at?
Denaturation. The double stranded DNA must be
denatured.
The denaturation step “unravels” the double helix, separating it into two strands.
◦ Denaturing DNA is done through
temperatures around 95 Celsius
What is the second steps in Polymerase Chain Reaction?
What temperature is the Annealing step done at?
What do primers attach to?
Annealing. This step is at a lower temperature than Denaturing step.
Annealing is done at around 55-65 degree celsius, depending on the primer makeup
Primers seek and attach to their
compliment sequence (target sequence)
What is the third step in PCR?
What is used to perform the visualizing step?
What else can be used to perform the visualizing step?
What shows real time amplification of DNA through detection systems withing the thermo cycler?
Visualizing
Mostly done with gel electrophorisis to
show length of sequence
Can also be done with fluorescence
rtPCR can show real time amplification of
DNA through detection systems within the
thermo cycler
What is In-situ hybridization?
Method that uses labeled complementary DNA or RNA to localize a specific DNA or RNA sequence in a section of tissue.
