Extraction and bisulfite conversion of the DNA from formalin-fixed
and paraffin embedded (FFPE) samples

 FFPE tumor and normal
samples from pancreatic cancer patients and FFPE normal samples from no
pancreatic cancer patients were collected. A pathologist confirmed the normal
sections from the hematoxylin and eosin (H & E) stained slides. Tissue
sections were serially cut as 10 mm thickness.  For extraction
DNA from FFPE samples, tissues are subjected to xylene treatment and rehydrated
using ethanol washes.  And then proteins are
digested by proteinase K and digestion lysis buffer, which contains denaturing
agents such as sodium dodecyl sulfate (SDS), at 55°C for 4 hours. Next,
bisulfite conversion was performed using Zymo EZ DNA Methylation Kit, Zymo
Research, following the manufacturer’s instructions.  Finally, bisulfite modified DNA was eluted by

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DNA Methylation Analysis

 Bisulfite treated DNA
samples were analyzed for the methylation levels of 3 genes, CDO1, TAC1 and
CHFR, using quantitative real-time Methylation Specific PCR (qMSP).  The qMSP levels were normalized against the respective
values of the internal control gene ?-Actin. The primers and hybridization
probes for qMSP were designed based on this sequence by using UCSC genomic
browser web site and Primer3 (v.0.4.0) 1.  Primer sequences
are listed in Table 1. Two ?l of bisulfited DNA was added to 23 ?l PCR mixture.

Final reaction condition was 1x buffer, 200 nM sense primer, 200 nM antisense
primer, 80 nM probe, 10 nM of fluorescein reference dye (Life Technologies),
0.167 mM dNTPs (VWRQuotation), and a single unit of Platinum Taq® DNA Polymerase
(invitrogen).  1x buffer consisted of 16.6mM
(NH4)2SO4, 67mM Tris
pH 8.8, 6.7 mM MgCl2 and 10mM -mercaptoethanol in a nuclease-free DI water
solution.  Amplification reactions were
performed using 96 well-plates (MicroAmp®) in
triplicate. Thermocycling conditions were: 95°C for 5 min, 50 cycles at 95°C
for 15 seconds, and 65°C for 1min and 72°C for 1 min.  The StepOnePlusTM
Real-Time PCR System was used (Applied Bio Systems).

Methylation values for each sample were calculated DCT method: DCT= CTsample – CT?-Actin. For replicates which were not detected, a CT of 100 was
used, creating minimum methylation value of near 0.  The mean 2^-DCT
value was calculated as follows: the methylation value = (2^-DCTrepricate1 + 2^-DCTrepricate2 + 2^-DCTrepricate3) / 3 1.  For the methylation
value which was greater than 1, the value of 1 was used, creating max
methylation value of 1.   

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