TITLE: ht-HiTE: High-Throughput DNA extraction from FFPE tissue
AUTHORS: Christoph Geisenberger

[DESCRIPTION]
This protocols outlines a high-throughput method for DNA extraction from FFPE material. The workflow has been automated on a Biomek i5 platform. 1 to 2 tissue scrolls (10 µm) or tissue macrodissected from glass slides is used as input and eluted in 60 µl after nucleic acid purification. Mean and median yield is 86.6 and 72.0 ng/µl (equivalent to 4.3 to 5.3 µg of total DNA). 

[STEPS]
SECTION: Preparation
1. Each well of a DNA lo-bind 96 deep-well plates is pre-loaded with 500 µl of mineral oil
SECTION: Preparation
2. From each FFPE block, 1 thin section (2 µM) and 2 to 4 thicker sections (10 µM) are placed on glass slides. Thin sections are stained with hematoxylin and eosin (H&E) and used to select and mark regions of interest. Corresponding areas of 10 µM unstained sections are macroscopically dissected using a sterile scalpel blade. The tissue fragments are placed into wells of a 96-well plate preloaded with mineral oil. 

Alternatively, whole tissue scrolls (10 µM) can be placed into a well without macrodissection. 

 

SECTION: Deparaffinization, Tissue Lysis and Decrosslinking
4. To each well, add 100 µl of the following reaction mix:

 

Seal the plate and incubate:
1 hour at 56°C
incubate overnight (16 to 24 hours) at 80°C
SECTION: Deparaffinization, Tissue Lysis and Decrosslinking
5. Optional: RNA Digestion

If downstream applications benefit from removing RNA, perform the following:
add 4 µl of RNAse A to each well 
seal the plate, mix by vortexing
centrifuge quickly to collect liquid at the bottom of the plate
incubate 2 mins at room temperature
SECTION: Deparaffinization, Tissue Lysis and Decrosslinking
3. The plate is sealed, placed into an pre-heated oven and incubated for 15 min at 56°C. Paraffin should be completely molten and dissolved.
SECTION: DNA Purification
5.2. Option 1: Column-based purification

prepare a 1:1 mix of Buffer AL and absolute Ethanol (400 µl per well + 10% extra)
add 400 µl of pre-mixed Buffer AL and ethanol to each sample
transfer 600 µl of sample with Buffer AL and ethanol onto a 96-well DNeasy column plate
centrifuge plate for 15 min at ≥ 6,000 g and discard the flowthrough
add 500 µl Buffer AW1 
centrifuge plate for 5 min at ≥ 6,000 g and discard the flowthrough
add 500 µl Buffer AW2
centrifuge plate for 15 min at ≥ 6,000 g and discard the flowthrough
centrifuge plate for 2 min at full speed to dry sample
place on new collection plate and add 60 µl of Buffer AE
centrifuge plate for 4 min at ≥ 6,000 g 


 

SECTION: DNA Purification
5.3. Option 2: Bead-based purification

add 80 µl of Ampure XP SPRI beads to each sample (0.8x ratio)
seal the plate and mix by vortexing
centrifuge for 30 s at 3000 g to collect liquid at the bottom of the well 
place plate on magnet and pellet beads on wall of the well
remove supernatant
add 200 µl of Ethanol (70%)
wait for 30 s, remove ethanol
repeat ethanol wash once
air-dry beads for 10 minutes or until dry at room temperature
add 60 µl of nuclease-free H2O, resuspend beads by pipetting and remove plate from magnet
incubate 10 min at room temperature
move plate to magnet, pellet beads
transfer the supernatant containing the DNA to a fresh DNA lo-bind plate
SECTION: Quality control
6. DNA quantification 
Option 1: measure concentrations using a dye-based method such as the Invitrogen Qubit platform
Option 2: use a spectrometry-based method (i.e. Nanodrop). Overestimation by the Nanodrop can be corrected, refer to the manuscript for further information. 

Size distribution
If downstream applications rely on high molecular weight DNA, running selected samples on a Bioanalyzer or TapeStation can be utilized for further quality control. 
SECTION: Transfer
5.1. Option 1: Column-based purification
transfer 100 µl of sample per well to a fresh deep-well plate (part of Qiagen DNA Blood & Tissue 96 kit)
adjust sample volumes to 200 µl using nuclease-free water

Opton 2: Bead-based purification
 transfer 100 µl of sample per well to a fresh shallow-well DNA lo-bind plate