Molecular Diagnostics

Question 1

What are pre-analytic variables?

Answer 1

All the events occurring before specimen analysis, that may affect molecular test results

Question 2

Why are pre-analytic variables important?

Answer 2

Because they can effect the integrity and purity of a sample and by extent the accuracy of test results

Question 3

What are some pre-analytic variables in molecular testing?

Answer 3

Surgical procedures (loss of oxygen, drugs, anesthesia)
Temperature
Fixative
Tool and work station cleanliness (DNA/RNA contamination)

Question 4

How to avoid DNA/NA contamination?

Answer 4

Use a dedicated microtome for molecular testing

1 blade per specimen

Sterile water or RNA-away to clean equipment and workstation

DNA/RNA free collection tubes

Question 5

Paraffin manual macrodissection

Answer 5

scalpel and microscope to remove ROI from paraffin block

Question 6

Paraffin manual microdissection

Answer 6

10uM sections on non-charged unbaked slides

Test slide is stained to identify ROI

ROI is outlined and manually scraped off

Question 7

Paraffin Laser microscope capture

Answer 7

.10uM sections on non-charged unbaked slides

Test slide is stained to identify ROI

individual cells can be identified and collected

Question 8

Paraffin scrolls or curls

Answer 8

4uM section for stain and ROI ID

QUality test the DNA present

10-30uM scrolls collected

Question 9

Paraffin cores

Answer 9

Biopsy punch of tissue is taken from block

Good for when the ROI is small

Core is frozen in LiN2 and powdered for isolation

Question 10

How to deparaffinize a sample for molecular diagnostics?

Answer 10

1. Cover tissue with xylene and mix
2. 50C oven for 3-5 min melts paraffin
3. Vortex while hot then centrifuge to pellet
4. Remove xylene/paraffin supernatant
5. repeat 1-4 until paraffin is completely reoved
6. Add 100% EtOH to remove xylene
7. Vortex, pellet, decant
8. Repeat 6-7 2x
9. Air dry pellet in dust free zone to remove fumes

Question 11

Steps in DNA extraction?

Answer 11

Protease digestion: changing digestion buffer daily. May take up to 2 days

DNA is reconstituted with buffer and a few uL is used for quality testing

Question 12

4 methods for documenting DNA extraction?

Answer 12

BioAnalyzer: run on a chip, provides both quality and purity, several samples at once

Spectrophotometers (NanoDrop): absorbance of light in UV spectrum
260(nucleic acid)/280(protein)
High ratio is better purity

Fluorometry: fluorescent dye in double stranded DNA is detected, less sensitive to impurities, more specific to double stranded DNA

Southern Blot: laborious and hazardous chemicals

Question 13

How should DNA be stored?

Answer 13

-80C for long term
-20C is also good/typical
4C for short term
NO THAW CYCLES

Question 14

3 techniques that utilize extracted DNA?

Answer 14

ISH
PCR
NGS

Question 15

ISH: In-Situ Hybridization

Answer 15

Uses DNA probes to quantify the number of gene copies in tissue sections

Question 16

PCR: Polymerase Chain Reaction

Answer 16

Amplifies small amounts of DNA exponentially

Good for small samples or investigating a specific gene of interest (expression)

Question 17

NGS: Next Gen Sequencing

Answer 17

Sequence the nucleotides in a section of DNA

Used in disease diagnosis