Scientific Posters

Zymo Research's scientific posters highlight the latest breakthroughs in molecular biology, showcasing innovations spanning from epigenetics to microbiomics. We are proud to present insights from our collaborations with leading scientists and institutions worldwide, with more contributions to the field of life science on the horizon.

Pluripotency Prediction Using a Key Set of Six Epigenetic Biomarkers
Jill Petrisko, Lam Nguyen, Manuel Krispin, Xi Yu Jia

The ability to accurately characterize the pluripotent state of human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells is paramount to the field of human stem cell research. Pluripotent stem cells display cell morphology, cell surface markers, a gene expression profile, and an epigenetic signature distinct from that of partially or fully differentiated cells. Routine measures employed to characterize the pluripotent state of an ES or iPS cell line include embryoid body formation, karyotyping, expression of transcription factors OCT4, SOX2, and NANOG, expression of specific cell surface antigen markers such as SSEA and TRA, and more recently, characterization of the cells' epigenetic profile. Genome-wide DNA methylation signatures for human ES and iPS cell lines have been studied using both reduced-representation bisulfite sequencing (RRBS) (Bock et al. 2010) and bead array platforms (Bibikova et al. 2006). While these genome-wide methods are the most accurate means for characterizing the epigenetic variability among ES and iPS cells lines, they are expensive and cumbersome for high-throughput research laboratories wanting to routinely monitor the quality of their pluripotent cell lines.

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DNA Methylation Markers for the Surveillance of Non-Muscle Invasive Bladder Cancer: Results from a Prospective Pilot Study
Paolo Piatti, Sanam Ladi-Seyedian, Sidney Roberts, Farshad Sheybaee Moghadam, Alireza Ghoreifi, Jeffrey Bhasin, Benjamin Jara, Lucy Sanossian, Yap Ching Chew, Sumeet Bhanvadia, Hooman Djaladat, Anne Schuckman, Gangning Liang, Siamak Daneshmand

Cystoscopy and urine cytology are routinely employed during follow-up of patients with a history of non-muscle invasive bladder cancer (NMIBC) due to the high recurrence rate of this disease. Diagnostic accuracy of FDA approved urine-based tests is suboptimal. Herein, we compare the diagnostic value of urine cytology and a newly developed urine-based DNA methylation test (Bladder CARE™) for surveillance of NMIBC

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Profiling DNA Methylation at Single-Nucleotide Resolution in Plasma Cell-free DNA Using Whole-Genome Bisulfite Sequencing (WGBS)
Yi Xu, Caila Ruiz, Hanjun Kim, Ryan Yancey, Zhenguo Zhang, Jeffrey Bhasin, Mingda Jin, Zhenfeng Liu, Angela Garibaldi, Xiaojing Yang, Larry Jia

Circulating cell-free DNA (cfDNA) has been reported to contain valuable genetic and epigenetic information for the diagnosis and prognosis of cancer. Studies have shown that the blood from cancer patients contained more tumor-derived cfDNA compared to the healthy controls. DNA methylation has been validated as one of the key drivers in the development of many diseases including cancer cfDNA methylation thus holds great potential to become a biomarker that will enable early detection of cancer. To evaluate the cancer detection values of cfDNA methylation markers, cfDNA was extracted from plasmas originated from patients with lung cancer and healthy controls using The Quick-cfDNA™ Serum & Plasma Kit, respectively. Whole-genome bisulfite sequencing (WGBS) was utilized to profile the global DNA methylation patterns between these two conditions.

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Quantitative Digital PCR Analysis of Cancer Gene Promoter Methylation Using Low Amounts of Input DNA
Paolo Piatti, Alireza Ghoreifi, Sanam Seyedian, Yap Ching Chew, Benjamin Jara, Lucy Sanossian, Jeffrey Bhasin, Michael Basin, Taikun Yamada, Gerhard Fuchs, Sumeet Bhanvadia, Rene Sotelo, Andrew Hung, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, Hooman Djalada

Silencing of tumor suppressor and tumor-related gene transcription by hypermethylation at promoter CpG motifs is a significant mechanism at work in human tumorgenesis. Various methods have been developed to analyze DNA methylation levels, both across the genome and at specific loci, in order to discover and interrogate disease relevant loci for methylation-based transcriptional control. Methods able to quantitatively measure differences in DNA methylation between normal and cancer cells provide promising sources for biomarker identification and assessment. Zymo Research's OneStep qMethyl™ Kit is used for the detection of region-specific DNA methylation via the selective amplification of methylated cytosines after digestion with methylation-sensitive restriction enzymes. RainDance Technologies' RainDrop™ dPCR system is a highly sensitive tool for precise quantification of nucleic acids using probe-based qPCR reagents. RainDrop offers unique analytical advantages for clinical diagnostics due to its exceptionally high sensitivity for absolute quantification and capability to multiplex assays, using a wide dynamic range of input DNA in a contamination-free and simple workflow. The combination of both technologies enables a novel, fast, and robust method for quantitative methylation analysis of small amounts of DNA. Here we report on the use of Zymo's methylation assay and RainDrop digital PCR counting to enable quantitative measurements of DNA methylation at specific genomic loci using small amounts of non-bisulfite converted DNA. MGMT promoter methylation measurements are used to illustrate the method's robust quantification of small percent methylation changes using low amounts of input DNA in a single-plex and duplex assay format (duplex RAB25-VIC and MGMT-FAM probes). Duplex assays using a methylation-independent reference assay and either RARB or CCND2 are used to assess promoter methylation in breast cancer tumor and adjacent normal tissue samples showing stage-specific differential methylation.

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Rapid Enzymatic DNA Degradation for Quantitation of 5-Methylcytosine and 5-Hydroxymethylcytosine
James L. Yen & Xi Yu Jia

Modification of DNA, namely 5- methylcytosine, has been recognized to be one of the dominant phenomenon in the field of epigenetics. Fluctuations in global DNA methylation levels have implications in development, cancer, and aging. Therefore methods for precise quantification of global DNA methylation (i.e. - HPLC and LC/MS) are powerful tools to gain a better understanding in these areas. To address the need for a rapid and convenient sample preparation method for global methylation quantitation, we have developed a one hour, one-step enzymatic procedure for DNA degradation with DNA Degradase™ and DNA Degradase Plus™. DNA Degradase™ and DNA Degradase Plus™ degrade DNA to single nucleotides and nucleosides, respectively. Nucleotides are easily quantitated by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC), while nucleosides (lacking a charged phosphate) are ideal for quantitation by mass spectrometry (LC/MS). Furthermore, we have validated DNA Degradase™ and DNA Degradase Plus™ by HPLC nand LC/MS, respectively. Sampling a range of biological sources, DNA Degradase Plus™ coupled with LC/MS has proven to be a powerful method for detection and quantification of 5-methylcytosine as well as 5-hydroxymethylcytosine.

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