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Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse.

Schillebeeckx M, Schrade A, Löbs AK, Pihlajoki M, Wilson DB, Mitra RD - Nucleic Acids Res. (2013)

Bottom Line: Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields.We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters.Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, 4444 Forest Park Parkway, St. Louis, Missouri 63110, USA.

ABSTRACT
DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.

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Related in: MedlinePlus

LCM-RRBS is reproducible and robust across 1 ng extracted from bulk fresh frozen and FFPE samples. CGI methylation (top panels) and the methylation at 2-kb regions flanking the TSS (bottom panels) were compared between (A) 1 ng (LCM-RRBS) and 400 ng of purified leukocyte genomic DNA (RRBS), and (B) 1 ng of FFPE DNA and 1 ng of fresh frozen genomic DNA extracted from the same endometrial tumor (LCM-RRBS).
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gkt230-F2: LCM-RRBS is reproducible and robust across 1 ng extracted from bulk fresh frozen and FFPE samples. CGI methylation (top panels) and the methylation at 2-kb regions flanking the TSS (bottom panels) were compared between (A) 1 ng (LCM-RRBS) and 400 ng of purified leukocyte genomic DNA (RRBS), and (B) 1 ng of FFPE DNA and 1 ng of fresh frozen genomic DNA extracted from the same endometrial tumor (LCM-RRBS).

Mentions: To evaluate the performance of LCM-RRBS, we benchmarked it against RRBS. Using 1 and 400 ng for LCM-RRBS and RRBS, respectively, we compared the genome-wide DNA methylation status of DNA isolated from human blood leukocytes. We did not do LCM on the 1 ng of purified DNA; instead, we only applied the downstream library preparation of the LCM-RRBS protocol (Figure 1). LCM-RRBS was able to interrogate >75% of CGIs and >65% of gene promoters, results that were similar to those obtained by RRBS (Supplementary Figure S1). LCM-RRBS was able to accurately measure the DNA methylation levels of CGIs and core promoters (Figure 2A) as well as individual CpG dinucleotides (Supplementary Figure S2). Increasing the required coverage for each CpG considered for CGI methylation did not significantly alter the concordance between RRBS and LCM-RRBS (Supplementary Figure S3). For CGIs (n = 18 448) and promoters (n = 8500) having at least 50 methylation measurements, we observed a Pearson correlation of 0.98 and 0.94, respectively, between 1 and 400 ng (Figure 2A). Most CGIs are either highly methylated (80–100%) or highly unmethylated (0–20%). We therefore sought to test how well LCM-RRBS could call a CGI as methylated or unmethylated. For CGIs with at least 50 high-quality CpG measurements, LCM-RRBS identified methylated CGIs with 91% sensitivity and 99% specificity and unmethylated CGIs with 97% sensitivity and 94% specificity when compared with the RRBS dataset. We therefore conclude that LCM-RRBS functions on as little as 1 ng of genomic DNA, interrogates most CGIs and promoters and is very accurate.Figure 2.


Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse.

Schillebeeckx M, Schrade A, Löbs AK, Pihlajoki M, Wilson DB, Mitra RD - Nucleic Acids Res. (2013)

LCM-RRBS is reproducible and robust across 1 ng extracted from bulk fresh frozen and FFPE samples. CGI methylation (top panels) and the methylation at 2-kb regions flanking the TSS (bottom panels) were compared between (A) 1 ng (LCM-RRBS) and 400 ng of purified leukocyte genomic DNA (RRBS), and (B) 1 ng of FFPE DNA and 1 ng of fresh frozen genomic DNA extracted from the same endometrial tumor (LCM-RRBS).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675465&req=5

gkt230-F2: LCM-RRBS is reproducible and robust across 1 ng extracted from bulk fresh frozen and FFPE samples. CGI methylation (top panels) and the methylation at 2-kb regions flanking the TSS (bottom panels) were compared between (A) 1 ng (LCM-RRBS) and 400 ng of purified leukocyte genomic DNA (RRBS), and (B) 1 ng of FFPE DNA and 1 ng of fresh frozen genomic DNA extracted from the same endometrial tumor (LCM-RRBS).
Mentions: To evaluate the performance of LCM-RRBS, we benchmarked it against RRBS. Using 1 and 400 ng for LCM-RRBS and RRBS, respectively, we compared the genome-wide DNA methylation status of DNA isolated from human blood leukocytes. We did not do LCM on the 1 ng of purified DNA; instead, we only applied the downstream library preparation of the LCM-RRBS protocol (Figure 1). LCM-RRBS was able to interrogate >75% of CGIs and >65% of gene promoters, results that were similar to those obtained by RRBS (Supplementary Figure S1). LCM-RRBS was able to accurately measure the DNA methylation levels of CGIs and core promoters (Figure 2A) as well as individual CpG dinucleotides (Supplementary Figure S2). Increasing the required coverage for each CpG considered for CGI methylation did not significantly alter the concordance between RRBS and LCM-RRBS (Supplementary Figure S3). For CGIs (n = 18 448) and promoters (n = 8500) having at least 50 methylation measurements, we observed a Pearson correlation of 0.98 and 0.94, respectively, between 1 and 400 ng (Figure 2A). Most CGIs are either highly methylated (80–100%) or highly unmethylated (0–20%). We therefore sought to test how well LCM-RRBS could call a CGI as methylated or unmethylated. For CGIs with at least 50 high-quality CpG measurements, LCM-RRBS identified methylated CGIs with 91% sensitivity and 99% specificity and unmethylated CGIs with 97% sensitivity and 94% specificity when compared with the RRBS dataset. We therefore conclude that LCM-RRBS functions on as little as 1 ng of genomic DNA, interrogates most CGIs and promoters and is very accurate.Figure 2.

Bottom Line: Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields.We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters.Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, 4444 Forest Park Parkway, St. Louis, Missouri 63110, USA.

ABSTRACT
DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine-guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.

Show MeSH
Related in: MedlinePlus