OMIC techniques on degraded tissue samples Pim French Dept - - PowerPoint PPT Presentation

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Apr 09, 2024 28 likes •324 views

OMIC techniques on degraded tissue samples Pim French Dept Neurology FFPE FFPE: formalin fixed, paraffin embedded The choice of storage for pathology labs FF tissues: 1000s FFPE tissues millions worldwide, ~20 million each

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OMIC techniques on degraded tissue samples

Pim French Dept Neurology

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FFPE

FFPE: formalin fixed, paraffin embedded
The choice of storage for pathology labs

– FF tissues: 1000’s – FFPE tissues millions worldwide, ~20 million each year in US – Well characterized samples

Histological
Pathological
Clinical
Clinical trial

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Formaldehyde

Formalin=Formaldehyde in solution
Crosslinks primary amino groups through CH2 linkage

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Primary amino group

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FFPE and DNA: Protein-DNA and interstrand crosslinks

Huang, J Am Chem Soc 1993; Lu, J. Am. Chem. Soc 2010

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FFPE fixation comes at a price

1. Degradation: Nicks and Double strand breaks 2. Hydrolysis of N-glycosyl bond 3. Deamination 4. Oxidation

1. 8-hydroxyguanine formation 2. Thymine or cytosine glycol formation

5. Thymine dimers

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Storage in FFPE compromizes DNA/RNA quality 1: Degradation

Formalin causes hydrolysis of

phosphodiester bonds

Dependent on time in paraffin

– (extra variable)

– 100 b fragments

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Storage in FFPE compromizes DNA/RNA quality 2: Hydrolysis of N-glycosyl bond

– Depurination: 10.000/cell/day – Depyrimidination: 500/cell/day

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Storage in FFPE compromizes DNA/RNA quality 3: Deamination

Rate ~100/cell/day

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Storage in FFPE compromizes DNA/RNA quality 4: Oxidation (Glycol formation)

Glycol: two hydroxyl (−OH) groups attached to

different carbon atoms.

Thymidine glycol: 100/cell/day

Chaw, Biochemistry 1980)

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Solving FFPE problem: 1: Adopt method (DNA)

Uracil-DNA glycosylase

Do, Oncotarget 2012, Clin Chem 2013

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Solving FFPE problem: Repair damage

Mono-methylol group additions (-CH2 OH) can be

reversed by heat

Enzymatic FFPE restoration

– mix of various DNA repair enzymes – cocktail of (proofreading) DNA polymerase, DNA repair enzyme and ligase

Arutyunyan, Izvestiya Akademii Nauk SSSR, Seriya Khimicheskiya, 1968.

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Problems working with FFPE tissue samples: RNA

RNA: Most arrays/methods are 3’ based and no

longer work

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Solving FFPE problem: Adopt method (RNA)

Revert chemical modification
Use random primers for cDNA synthesis
But:

– only mRNA has poly A tail – mRNA is only ~2% of all RNA in a cell

Requires rRNA depletion

– Ribominus – Ribo zero

LNA to rRNA specific sequences

mRNA: proteins (~2%) rRNA: ribosomes (~80%) tRNA: codons (~15%)

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Examples of high throughput methods using FFPE tissues

Array based

– DNA

Oncoscan DX (Affymetrix)
Methylation arrays (Illumina)

– RNA

Exon arrays (Affymetrix)
DASL (Illumina)
Sequencing

– DNA – RNA

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DNA-arrays: Oncoscan

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DNA methylation

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DNA methylation on FFPE tissues

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Molecular diagnostics on FFPE

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Targeted sequencing: PCR

Small PCR amplicons

Histology Molecular

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Targeted sequencing: Baits

Illumina TruSeq, Agilent Sureselect, Roche/Nimblegen etc

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Exome sequencing: Mutations

Gad Getz, Kristin Ardlie

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Exome sequencing: CNV

Gad Getz, Kristin Ardlie

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RNA expression: Exon array

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RNA-Seq

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Agilent sureselect

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Conclusions

Analysis of FFPE tissues is feasible

(RNA/DNA/Methylation)

Be aware of noise in the data