DNA EXTRACTION FROM MICRODISSECTED PARAFFIN SECTIONS
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DNA EXTRACTION FROM MICRODISSECTED PARAFFIN SECTIONS
This is a four day procedure so it's best to start on Monday or Tuesday.
H&E stained thin sections are first reviewed by a pathologist, and areas of interest are outlined. Tissue blocks are then cut as follows:
3 consecutive 5-micron sections from a formalin fixed paraffin embedded block are cut and placed onto positively charged slides (some dissection protocols discourage use of charged slides). More can be cut if additional studies (e.g. immunohistochemistry) are being considered.
Methyl Green (see protocol below).
Day 1:
DISSECTION:
See photos of H&E , methyl green before dissection , and methyl green after dissection .
Label the top of 0.5 ml pcr tubes, color code dot label them also.
Add 15 ul of 1X PCR/PK Buffer into each tube, close caps.
Do one case at a time. If normal is being dissected do this first.
Scrape away areas that you dont want away from the sample (use a #11 blade).
Place this carefully into the 0.5 ml pcr tube with the pcr/pk buffer.
Go onto next area. Remember to use diiferent blades for each part of the tumor.
Overlay with 20 ul mineral oil. Close cap and seal with parafilm.
DIGESTION:
Incubate overnight for shaking at 120 rpm, 55 C.
Day 2:
Day 3:
Day 4:
Remove oil by rolling samples and oil on parafilm and and pipette aqueous DNA into new tube.
SOLUTIONS
Digestion buffer: 1X PCR Buffer with 0.5% Tween20 / 0.4mg/ml pk stored in aliquots at -20 C.
PK: Stored as 20mg/ml aliquots at -20 C; can be refrozen a few times.
Methyl Green Staining
Methyl Green is used for staining of microdissected slides since we have found that hematoxylin (H&E) can interfere with PCR amplification. It is usually only necessary to stain and microdissect one slide, but two slides are used when the target is less than 1 mm in diameter.
- Xylene 2X 3 mins each.
- 100% ethanol 2 mins.
- 95% ethanol 2 mins.
- 70% ethanol 2 mins.
- 50% ethanol 2 mins.
- dH2O 2 mins.
- 0.1-1% Methyl green (dip 3-6 times or leave ~15-60 seconds).
- The section should not be too dark. The methyl green concentration can be varied if sections are generally too dark or too light. It's better to start with a lower concentration.
- dH2O 1-5 mins (depending on how blue/green the tissue is).
- Air dry upright.
- The slide is now ready for photographing and microdissection. It is better to use soon but can be stored for a few weeks if necessary.
