DNA EXTRACTION FROM MICRODISSECTED PARAFFIN SECTIONS
This is a four day procedure so it's best to start on Monday or Tuesday.
CASE SELECTION:
H&E stained thin sections are first reviewed by a pathologist, and areas of interest are outlined. Tissue blocks are then cut as follows:
Three to Nine 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.
Stain the middle slide with H&E. Check that areas from the previous H&E slide are the same and if needed get the pathologist to review the new H&E slide (some small lesions will change diagnosis or even disappear in deeper sections). If the two H&E's look the same then Stain one or both with Methyl Green (see protocol below).
If the histopathology is at all difficult, photos are taken of areas to be dissected on both the methyl green and H&E slides. In general, the methyl green slide is photographed at a higher power than the H&E.
The pathologist then reviews the methyl green photo along side of the H&E slide. The methyl green photo is then marked by the pathologist to define the areas to be microdissected. If additional areas with no photos are suggested for use, mark the H&E slide, then take photos while dissecting to document what is taken. If there is any uncertainty, ask the Pathologist to re-review the area on the slide in question.
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.
For small tumors Add 15 ul of 1X PCR/PK Buffer into each tube, close caps. For larger tumors add 100 ul.
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).
Take a new #15 blade and dip (don't dunk) tip of blade into the labeled tube with pcr pk buffer, put this very small drop from the tip of blade onto the sample area by tapping.
Start scraping tissue into the center of puddle. Watch puddle dry up to point where you can pick up the sample scrapings (do not overdry or tissue will fly away!!).
Place this carefully into the 0.5 ml pcr tube with the pcr/pk buffer. For
larger tumors you may want to start with a 1.5 ml tube
The amount of pcr/pk buffer depends on the sample. In general it's 15 ul for anything less than 1 mm, and proportionally more if larger.
Go onto next area. Remember to use different blades for each part of the tumor.
For volumes less than 100 ul overlay with 20 ul mineral oil. Close cap and seal with parafilm. For larger volumes oil is not necessary.
DIGESTION:
Incubate overnight for shaking at 120 rpm, 55 C.
Day 2:
Add 0.3 ul fresh conc Proteinase K (20 mg/ml stock) through the oil to sample. (It's 0.3 ul per 15ul original volume, so adjust as necessary).
Incubate overnight at 55 C.
Day 3:
(Repeat:) Add 0.3 ul fresh conc Proteinase K (20 mg/ml stock) through the oil to sample. (It's 0.3 ul per 15 ul original volume, so adjust as necessary).
Incubate overnight at 55 C.
Day 4:
Remove all parafilm from tubes. Inactivate Proteinase K for 10-15 minutes at 95 C in PCR machine or hot water bath.
Remove oil by rolling samples and oil on parafilm and and pipette aqueous DNA into new tube.
DNA Purification and Buffer Exchange
We use Amicon Microcon YM-30 columns (made by Millipore) to concentrate the DNA. We follow the protocol provided with the kit.
Briefly:
- Add 450 ul ddH20 to column, spin 8 minutes at 12,000 x g at room temperature.
ˇ Discard flow through.
ˇ Add water to the DNA to be concentrated up to a volume of 450 ul.
ˇ Apply to column, spin 8 mins. at 12,000 x g at room temperature.
ˇ Discard flow through.
ˇ Add 450 ul ddH20 to column, spin 8 mins at 12,000 x g at room temperature.
ˇ Discard flow through.
ˇ Add 450 ul ddH20 to column, spin 8 mins at 12,000 x g at room temperature.
ˇ
Dump flow through. Check for amount of water remaining
on top of the column.
If it looks like more than 15 ul, then spin column again for 2 more minutes.
(This step is critical, as the spinning time controls the amount of solution
the DNA will be in).
ˇ Flip the column into a new 1.5 ml tube. Spin 1 minute at 13,000 x g. This is your DNA. The output volume can range from 10 ul to 100 ul.
Taqman
Quantitation of DNA
The amicon column purified DNA from microdissected tissue is measured quantitatively by Taqman using a CA-repeat fluorogenic probe as per “Ginzinger, D.G., et al. Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis. Cancer Res, 2000. 60(19): p. 5405-9”. The probe set used is referred to as human pool 2 and includes probes for D1S2868 (1p22), D2S385 (2q31), D4S1605 (4p16), D5S643 (5q32), D10S586 (10p12), and D11S1315 (11p15). These loci did not usually show alterations in human ovarian tumors.
Sample DNA set up.
- Genomic DNA (cat# G147 Promega), measured by fluorometer, diluted to 5 ng/ul
- Serial dilutions of genomic DNA at 30, 3, 0.3 and 0.03 ng/ul, enough to run in duplicate (see below)
- Wipe down a PCR area and a separate “DNA” bench with 70% ethanol
- Use filtered pipet tips. NOTE: accurate pipetting is critical.
- In a pcr clean room take an optical 96 well plate, (MicroAmp Optical 96-well Reaction Plate, PE Applied Biosystems) with 8 wells empty for the duplicate standard curve, pipet 4 ul nuclease free water into remaining wells using a multichannel pipettor, cover with strip caps.
- After the water has been added and the wells have been covered, take the plate elsewhere to add the DNA.
- Add 1 ul of DNA per well. In general we use 1ul of microdissected paraffin DNA, and 1 ul of fluorometry measured “fresh” DNA diluted to 5 ng/ul.
Set-up of
Quantitative DNA measurement by TaqMan
ˇ
This
part of the protocol was kindly Provided by the UCSF-Genome Analysis Core
facility
Contacts:
Mamie Yu 502-1861 & David Ginzinger 476-3665
Master
Mix for DNA
Quantification
ˇ
The amount
of master mix you need to make will vary depending on the number of reactions
you want to run.
ˇ
The probe used is a CA-repeat fluorogenic probe consisting of
5’-FAM-TGT-GTG-TGT-GTG-TGT-GTG-TGT-6-carboxytetramethylrhodamine-3’
- The Reference primer set used is referred to as human pool 2 and
includes probes for D1S2868 (1p22), D2S385 (2q31), D4S1605 (4p16), D5S643
(5q32), D10S586 (10p12), and D11S1315 (11p15). These loci did not usually show alterations in human
ovarian tumors.
ea
(ul) Final
Conc
5x Taqman Buffer 10 1x
25 mM MgCl2 11 5.5
mM
25 mM dNTPs 0.4 200
uM
dH2O 15.45
8.33 uM Ref pool primers 2.4 400
nM
5 U/uL AmpliTaq 0.25 1.25
U
20 uM CA (12.5) probe 0.5 200
nM
40 mix
final volume
Standard
Curve
This is
prepared as a 10 Fold Serial Dilution of DNA (3ng/ul starting DNA), 4 Pts
The standard
curve is run in duplicate with 4 concentrations.
For each subsequent
aliquot use previous concentration
DNA(ul) dH2O
(ul) Total
Vol(ul) 10 ul =
25 0 25 30ng/rxn. Use
10ul /well in duplicate
2.5 22.5 25 3.0ng/rxn. Use
10ul /well in duplicate
2.5 22.5 25 0.3ng/rxn. Use
10ul /well in duplicate
2.5 22.5 25 0.03ng/rxn. Use 10ul
/well in duplicate
Add 10ul of
serial diluted DNA of each known concentration
ˇ
Samples for
quantification: see above
Sample to be
submitted by user: 1ul sample + 4ul dH2O. Add 5ul dH2O per sample.
ˇ
Quick
centrifuge of plate to spin down any sample.
ˇ
Add 40ul
master mix per well.
ˇ
Run plate on
Instrument: ABI 7700
Thermal Cycler Conditions: Stage1-
95ēC 12m,
Stage 2- 95ēC 15s, 60ēC 1m for 40cycles
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.
A. Deparaffinization:
- Xylene 2X 3 mins each.
- 100% ethanol 2 mins.
- 95% ethanol 2 mins.
- 70% ethanol 2 mins.
- 50% ethanol 2 mins.
- dH2O 2 mins.
B. Staining:
- 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. Tissue which have a lot of epithelial cells will stain darker than tissue with stromal tissue.
- 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.
S. DeVries / F. Waldman
Email questions to Sandy DeVries at the UCSF Cancer Center (devries@cc.ucsf.edu)