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| Epigenetics
Applied Biosystems
Methylation Analysis
850 Lincoln Centre Drive
Foster City, CA 94404
800.327.3002 / 650.638.5800
http://www.appliedbiosystems.com
prodinfo@appliedbiosystems.com
Methylation Analysis Using FFPE Samples: A Step-by-Step Guide
Aberrant DNA methylation patterns have been implicated in many
STEP 4
disease systems, including cancer. Bisulfi te sequencing is the gold
Detection and Analysis
standard in assessing the methylation status of a particular region
Amplifi ed products from step 3 are sequenced with the BigDye
®

because only sequencing provides direct detection of methylation
Terminator v1.1 Cycle Sequencing Kit and M13-tailed primers,
events, as well as information across the entire amplifi ed region.
using a slightly modifi ed thermal cycler profi le. Bisulfi te sequencing
When working with clinical samples, researchers often need to
reactions are purifi ed using the BigDye XTerminator
TM
Purifi cation
use DNA that has been stored as formaldehyde-fi xed paraffi n-
Kit, which removes unincorporated BigDye Terminators quickly and
embedded (FFPE) tissue blocks. DNA from FFPE tissue blocks is
easily. The reactions are then run on a 3130 Genetic Analyzer.
diffi cult to analyze due to varying degrees of fragmentation and
protein contamination. This protocol describes how easy it is to
A Complete Solution
generate high quality methylation results from DNA isolated from
Applied Biosystems provides a streamlined workfl ow for bisulfi te
FFPE samples.
sequencing that takes you from DNA isolation to fi nal results, with
optimized protocols for each step.
STEP 1
DNA Isolation from FFPE Tissues
For more information, visit
DNA is isolated using the RecoverAll
TM
Total Nucleic Acid Isolation
http://info.appliedbiosystems.com/methylation
Kit, which provides high quality, high-molecular-weight DNA, free
from PCR inhibitors. RecoverAll kits are optimized to release a
Reference
maximal amount of DNA fragments of all sizes, with typical yields of
1. DNA methylation and cancer. Das P.M., Singal R. J. Clin. Oncol., 2004,
more than 50% that of DNA recovered from unfi xed tissue from the 22:4632–42.
same sample source.
2. Bisulfite conversion of genomic DNA for methylation analysis: protocol
STEP 2
simplification with higher recovery applicable to limited samples and
Bisulfi te Conversion
increased throughput. Boyd, et al. Anal. Biochem., 2004, Mar 15;
326(2):278–80.
DNA is converted using the methylSEQr
TM
Bisulfi te Conversion Kit.
This kit uses size exclusion column purifi cation, which increases
3. Recovery of bisulfite-converted genomic sequences in the methylation-
sample yields and reduces the amount of bias seen with resin
sensitive QPCR. Munson, et al. Nucleic Acids Research, 2007,
purifi cation methods. Unbiased DNA recovery of methylated and 35(9):2893–903.
unmethylated fragments enables more accurate downstream
quantitative analysis. The methylSEQr kit uses gentle denaturation
conditions that reduce DNA fragmentation and increase the amount
of high quality DNA available for amplifi cation.
STEP 3
Assay Design and Amplifi cation
Primers are designed using Methyl Primer Express
®
Software v1.0,
a free online primer design tool specifi cally for methylation studies.
The software performs an in silico bisulfi te conversion (Cs are
converted to Ts) and aids in the selection of primers.
Amplifi cation of the bisulfi te-converted DNA from step 2 is performed
using AmpliTaq Gold
®
DNA Polymerase on Applied Biosystems
thermal cyclers, including the Veriti
TM
96-Well Fast Thermal Cycler
(in standard ramp mode). The PCR product is purifi ed enzymatically
with ExoSAPIT
®
Reagent (USB Corporation).
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 23
AppliedBioSys Protocol.indd 23 10/25/07 6:56:44 PM
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