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Epigenetics of the Placenta
In light of the above, we have undertaken a comprehensive remove single stranded DNA. Reactions were incubated for 30 min
analysis of cytosine methylation patterns in chorionic villus at 37uC and inactivated at 65uC for 20 min. Reactions were then
samples (CVS) and gestational age-matched maternal blood cells phenol-chloroform extracted and the DNA precipitated and
(MBCs) using two distinct microarray based methods. We provide resuspended in 21.2 mL nuclease-free de-ionized water. Finally,
the first detailed analysis of the chorionic villus methylome at the extracted genomic DNA was quantified and assessed for purity
maternal interface in the context of both global gene expression using a NanoDrop ND-1000 UV-VIS Spectrophotometer.
patterns and primary DNA sequence.
CGH Target Labeling and Hybridization for Agilent
Materials and Methods Microarrays
Tissue Handling and DNA Extraction Experimental and reference DNA were labeled with Cy3-dUTP
The collection of tissue samples was approved by the University of and Cy5-dUTP respectively, and vice versa for dye-swaps, using a
BioPrime CGH Genomic Labeling kit per the manufacturer’s
Pittsburgh Institutional Review Board (PRO07070298). This project protocol (Agilent). Hybridization was performed in a mix
includes no involvement of human subjects according to the federal containing 50 mL of human Cot-1, 52 mL of Agilent 10x blocking
regulations [146.102(f)]. That is, no data was obtained through agent, 260 mL of Agilent 2x HiRPM hybridization buffer, and
intervention or interaction with the individual, nor was any 158 mL of the labeled DNA. The hybridization mix was heated to
identifiable private information obtained. All samples used in this 95uC for 3 minutes, then incubated at 37uC for 30 minutes and
study were discarded de-identified tissues. CVS samples were applied onto the active array area. Hybridization with gentle
obtained between gestational weeks 11 and 13 from the Magee agitation was carried out at 65uC for 40 hours. After hybridiza-
Womens Hospital Cytogenetic Screening Laboratory. All samples we tion, the slides were washed in Oligo aCGH Wash Buffer 1 and
confirmed to have normal karyotypes using standard cytogenetic Oligo aCGH Wash Buffer 2, followed by acetonitrile and
techniques. Samples were dissected under a microscope and Stabilization and Drying Solution (Agilent) per the manufacturer’s
separated from any decidual tissue or flecks of blood. The culture protocol. The slides were scanned using an Agilent Scanner and
media was removed and the tissue placed in 1.5–2.0 mL micro the data was analyzed using Agilent Feature Extraction software
centrifuge tubes before freezing at 280uC until DNA was extracted. 8.1 (Agilent). Visualization and comparison of the datasets were
To extract DNA, one 5 mm stainless steel bead and 180 mL buffer done with CGH-Analytics 3.2 (Agilent).
ATL (from Qiagen’s DNeasy Blood and Tissue kit) were added to
each CVS sample. The samples were placed in the TissueLyser Infinium Microarray Analysis
(Qiagen) Adaptor set 2624, and the TissueLyser was operated for 20 The Infinium data is MIAME compliant has been deposited in
seconds at 30 Hz. The DNA was then purified using the DNeasy
Blood and Tissue kit as per the manufacturer’s protocol. MBCs were the GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the
obtained between gestational weeks 11 and 13 from the Magee accession number/series record GSE23311. The HumanMethyla-
Women’s Hospital Prenatal Screening lab. DNA was extracted from tion27 DNA Analysis BeadChip (Illumina) allows interrogation of
the MBC’s using a modified protocol previously described by 27,578 CpG sites based on the NCBI CCDS database (Genome
Iovannisci, et al., 2006 [22], using reagents from the MasturePure Build 36) and also targets the promoter regions of 110 miRNA
DNA Purification Kit (Epincentre Technologies, Madison, WI, Cat. genes. Bisulphite conversion of DNA was carried out using the EZ
No. MCD85201). Briefly, clotted blood (approximately 1 mL) was DNA MethylationTM Kit (Zymo Research Corp., CA) to convert
mixed with an equal volume (1 mL) of 2X Tissue and Cell Lysis unmethylated cytosine nucleotides to uracil. Following denatur-
Solution, votexed for 10 s and combined with 2 mL Tissue and Cell ation with 0.1N NaOH, converted DNA samples were amplified
Lysis Solution (MasturePure kit) containing 25 ng/mL proteinase K. by incubation at 37uC for 20 hours in a proprietary amplification
2 mL of MPC Protein Precipitation Reagent was added to the total reaction mix. Amplified DNA was fragmented using vendor-
volume (4 mL) of the lysed sample and vortex vigorously for 10– supplied reagents by incubation for one hour at 37uC. Fragmented
15 sec, after which samples were cooled on ice for $1 hour. Cell DNA sample was precipitated and resuspended in hybridization
debris were then pelleted by centrifugation (x2) for at least 30 min at buffer. Infinium BeadChips were cleaned and activated by
$2000 g and supernatants transferred to new 50 mL conical tubes. washing with ethanol, formamide and vendor supplied pre-
DNA was precipitated in 2 volumes of isopropanol, purified by hybridization buffers. DNA samples are denatured, applied to
phenol/chloroform extraction and resuspended in 50 mL DNAse/ the Infinium arrays and hybridized 16–24 hours with rocking at
RNAse free water. 48uC. The BeadChip is placed into a flow-through chamber,
unhybridized and non-specifically hybridized DNA was washed
Target DNA Preparation for Agilent Microarray Analysis away and single base extension was performed on bound primers
The Agilent data is MIAME compliant has been deposited in the with labeled nucleotides. Hybridized DNA sample was removed
by washing using proprietary buffers. Staining steps were
GEO database (http://www.ncbi.nlm.nih.gov/geo/) with the performed to attach florescent dyes to the labeled nucleotides
accession number/series record GSE23835. Genomic DNA and the array surface sealed to protect the dyes from atmospheric
samples (3 mg) were digested for two hours at 37uC with 50U degradation. The final array was scanned using an Illumina
HpaII (New England Biolabs [NEB]) in 90 mL total reaction volume BeadArray Reader and the data analyzed using Bead Studio 2.0.
using NEB buffer 4. A second aliquot of 50U, 1 mL of buffer 4, and
4 mL water were added and digestion continued overnight (total Determination of the methylation status of CpG sites
reaction volume was 100 mL). Mock digestion controls were using Infinium Array Data
included to monitor digestion efficiency. Following overnight
digestion, reactions were digested further with 5 uL (50U) of TspRI On an Infinium array, each targeted CpG site was interrogated
(NEB) at 65uC for three hours. Reactions were then incubated by 2 probes: probe A for unmethylation status, and probe B for
further with 75U (0.75 ml) Exonuclease III (NEB) and incubated at methylation status. The A probe signals and B probe signals were
30uC for 1 hour. Enzymatic activity was then nullified by heating at normalized separately, using the cyclic loess algorithm (Wu). We
70uC for 20 min after which 50U of RecJF (NEB) were added to then computed the log ratio of probe B to probe A: log(B/A), as
PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e14723
