Pyrosequencing

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The Murphy lab has two Pyromark Q96 MD pyrosequencing instruments and can assist with quantitative analysis of DNA methylation using bisulfite pyrosequencing.  We are happy to consult with you to discuss your methylation project.  The Murphy lab has been using pyrosequencing, considered the gold standard for targeted analysis of DNA methylation, for over seven years to study numerous loci in humans and rodents. Pyrosequencing is a sequence by-synthesis approach which relies on input of a specific sequence to analyze. For methylation analysis, the genomic DNA is pre-treated with sodium bisulfite, a chemical that converts any unmethylated cytosine to uracils. Methylated cytosines are protected from this conversion.  Following PCR amplification of the bisulfite-modified DNA, pyrosequencing is used to determine the proportion of the sample analyzed that is methylated (cytosine retained) and unmethylated (appearing as thymines) at all potentially methylated cytosine positions within the sequence. We have published a methods paper that explains this in more detail.

We have previously published the ability of pyrosequencing to distinguish 5% differences in methylation, using mixtures of fully methylated and unmethylated sequences.  More recently, we have also shown that pyrosequencing is able to distinguish 0.5% differences in methylation.  Pyrosequencing is also an excellent tool to quantify allele-specific expression.  

Some of our relevant publications are listed below. For further information, please contact Susan Murphy or Zhiqing Huang.


References for pyrosequencing

Yamaguchi, K., Huang, Z., Matsumura, N., Mandai, M., Okamoto, T., Baba, T., Konishi, I., Berchuck, A. and S.K. Murphy. Epigenetic determinants of ovarian clear cell carcinoma biology. International Journal of Cancer (in press). PDF

Soubry, A., Murphy, S.K., Wang, F., Huang, Z., Vidal, A.C., Fuemmeler, B.F., Kurtzberg, J., Murtha, A., Jirtle, R.L., Schildkraut, J.M. and C. Hoyo. 2013. Newborns of obese parents have altered DNA methylation patterns at imprinted genes. International Journal of Obesity (in press).  PDF

Murphy, S.K., Yang, H., Moylan, C.A., Pang, H., Dellinger, A. Abdelmalek, M.F., Garrett, M.E., Ashley-Koch, A., Suzuki, A., Tillmann, H.L. Hauser, M.A. and A.M. Diehl. 2013. Relationship between the methylome and transcriptome in patients with non-alcoholic fatty liver disease. Gastroenterology 145:1076-1087.  PDF

Huang, Z. and S.K. Murphy. 2013. Increased intragenic IGF2 methylation is associated with repression of insulator activity and elevated expression in serous ovarian carcinoma. Frontiers in Women's Cancer 3:131.  PDF

Liu, Y., Hoyo, C., Murphy, S.K., Huang, Z., Overcash, F., Thompson, J., Brown, H. and A.P. Murtha. 2013. DNA methylation at imprint regulatory regions in preterm birth and infection. American Journal of Obstetrics and Gynecology 208:395.e1-7.  PDF

Vidal, A., Henry, N.M., Murphy, S.K., Oneko, O., Nye, M., Bartlett, J.A., Overcash, F., Huang, Z., Wang, F., Mlay, P., Obure, J., Smith, J., Vasquez, B., Swai, B., Hernandez, B. and C. Hoyo. 2013. PEG1/MEST and IGF2 DNA methylation in CIN and cervical cancer. Clinical and Translational Oncology (in press).  PDF

Vidal, A.C., Murphy, S.K., Murtha, A.P., Schildkraut, J.M., Soubry, A., Huang, Z., Neelon, S.E., Fuemmeler, B., Iversen, E., Wang, F., Kurtzberg, J., Jirtle, R.L. and C. Hoyo. 2013 Associations between antibiotic exposure during pregnancy, birth weight and aberrant methylation at imprinted genes among offspring. International Journal of Obesity 37:907-913.  PDF

Nye, M.D., Hoyo, C., Huang, Z., Vidal, A.C., Wang, F., Overcash, F., Smith, J.S., Vasquez, B., Hernandez, B., Swai, B., Oneko, O., Mlay, P., Obure, J. Gammon, M.D., Bartlett, J.A. and S.K. Murphy. 2013. Associations between methylation of Paternally Expressed Gene 3 (PEG3), cervical intraepithelial neoplasia and invasive cervical cancer. PLoS One 8:e56325.  PDF

Soubry, A., Schildkraut, J.S., Murtha, A., Wang, F., Huang, Z., Bernal, A., Kurtzberg, J., Jirtle, R.L., Murphy, S.K. and C. Hoyo. 2013. Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newbron Epigenetics STudy (NEST) cohort. BMC Medicine 11:29. PDF

Murphy, S.K. Bassil, C.F. and Z. Huang. 2013. Main principles and outcomes of DNA methylation analysis.  In: Methods in Molecular Biology, (Anastasia Malek, ed.), Humana Press (Springer) 1049:67-74.  PDF

Bassil, C.F., Huang, Z. and S.K. Murphy. 2013. Bisulfite pyrosequencing. In: Methods in Molecular Biology, (Anastasia Malek, ed.), Humana Press (Springer) 1049:95-107.  PDF

Joubert, B.R. Haberg, S.E., Nilsen, R.M., Wang, X., Vollset, S.E., Murphy, S.K., Huang, Z., Hoyo, C., Midttun, O., Cupul-Uicab, L.A., Ueland, P.M., Wu, M.C., Nystad, W., Bell, D.A., Peddada, S.D. and S.J. London. 2012. 450k Epigenome-wide scan identifies differential DNA methylation in newborns related to maternal smoking during pregnancy. Environmental Health Perspectives 120:1425-1431.  PDF

Murphy, S.K., Huang, Z. and C. Hoyo. 2012. Differentially methylated regions of imprinted genes in prenatal, perinatal and postnatal human tissues. PLoS One 7:e40924.  PDF

Liu, Y., Murphy, S.K., Murtha, A.P., Fuemmeler, B.F., Schildkraut, J., Huang, Z., Overcash, F., Kurtzberg, J., Jirtle, R., Iversen, E.S., Forman, M.R. and C. Hoyo. 2012. Depression in pregnancy, infant birth weight and DNA methylation of imprint regulatory elements. Epigenetics 7:735-746.  PDF

Hoyo, C., Fortner, K., Murtha, A.P., Schildkraut, J.M., Soubry, A., Demark-Wahnefried, W., Jirtle, R.L., Kurtzberg, J., Forman, M.R., Overcash, F., Huang, Z. and S.K. Murphy. 2012. Association of cord blood methylation fractions at imprinted insulin-like growth factor 2 (IGF2), plasma IGF2 and birth weight. Cancer Causes Control 23:635-645.  PDF

Perkins, E., Murphy, S.K., Murtha, A.P., Schildkraut, J., Jirtle, R.L., Demark-Wahnefried, W., Forman, M.R., Kurtzberg, J., Overcash, F., Huang, Z., and C. Hoyo. 2012. Insulin-like growth factor 2/H19 methylation at birth and risk of overweight and obesity in children. Journal of Pediatrics 161:31-39.  PDF

Murphy, S.K., Adigun, A., Huang, Z., Overcash, F., Wang, F., Jirtle, R.L., Schildkraut, J.S., Murtha, A.P., Iversen, E.S. and C. Hoyo. 2012. Gender-specific methylation differences in relation to prenatal exposure to cigarette smoke. Gene 494:36-43.  PDF

Hoyo, C., Murtha, A.P., Schildkraut, J.M., Jirtle, R.L., Demark-Wahnefried, W., Forman, M.R. Iversen, E.S., Kurtzberg, J., Overcash, F., Huang, Z. and S.K. Murphy. 2011. Methylation variation at IGF2 differentially methylated regions and maternal folic acid use before and during pregnancy. Epigenetics 6:928-936. PDF









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