DNA extraction, Polymerase Chain Reaction, and Sequencing : Workshop in Clinical Genetics

AbstractDNA extraction, Polymerase Chain Reaction (PCR), and Sequencing are basic methods in molecular biology and genetics. Those there are routinely performed as basic methods in genetic research and currently also for diagnostic lab especially for pathology and human genetics. With the advance in the genetics and clinical service for cancer management, mutation analysis is very important not only for diagnosis but also for prediction of therapeutic response. Detection of KRAS, BRAF, EGFR, and c-KIT mutations is presently performed in almost every molecular pathology lab as part of daily clinical service in cancer management. In this workshop we will discuss tips and tricks for those three basic lab methods. How to improve amount and purity of DNA extraction from blood and tissues, how to avoid DNA degradation during the procedure and storage, how to perform PCR, factors and substance that inhibit polymerases during PCR, how to design effective primer pairs, and how basic theory for sequencing, and interpretation of sequencing will be discussed. Although it has been widely discussed, this workshop is especially important for clinicians who previous do not have hands-on laboratory experience. In addition, number of labs with ability to perform and serve basic genetic and molecular analysis are still limited in Indonesia. With this workshop, we expect to improve knowledge and skill in DNA extraction, PCR, and Sequencing.Keywords : DNA, PCR, sequencin

DNA methylation and expression of Homeobox gene family as diagnostic and prognostic markers in human hepatocellular carcinoma

ABSTRACTHomeobox genes consist of a family of evolutionarily conserved genes that play important roles in morphogenesis, embryogenesis, and cell fate determination. Re-expression of embryogenic genes has been associated with carcinogenesis of human cancers. Aberrant expression of homeobox genes has been increasingly found to modulate diverse processes such as cell proliferation, cell death, metastasis, angiogenesis and DNA repair. We studied DNA methylation and expression of homeobox gene family in human hepatocellular carcinoma (HCC), the fifth most common cancer and the third leading cause of cancer mortality worldwide. We performed microarray for comprehensive DNA methylation and gene expression using primary HCC samples and healthy liver tissues. Confirmation using pyrosequencing and RT-PCR was then performed. Clustering both unsupervised and supervised methods using Qlucore software was then performed. Enrichment of homeobox genes both for DNA methylation and gene expression could differentiate HCC and the healthy liver tissues. Profile of homeobox gene methylation could further predict clinical outcome. Inverse correlation between DNA methylation and gene expression was shown (HOXA9, Spearman r=-0.49, p=0.002). Gain of DNA methylation in HOXA9, HOXA13, and MEOX1 correlated with shorter HCC survival (log-rank Mantel-Cox test p=0.02, with median survival 50 and 490 weeks, respectively). We demonstrated potential roles of DNA methylation and gene expression profiles of Homeobox gene family as diagnostic and prognostic marker in patients with HCC

PENDUGAAN NILAI HERITABILITAS UKURAN TUBUH PADA UMUR SAPIH DAN UMUR SETAHUN SAPI BALI DI BALAI PEMBIBITAN TERNAK UNGGUL SAPI BALI, JEMBRANA, BALI

Tujuan dari penelitian adalah untuk mengetahui nilai heritabilitas ukuran tubuh meliputi panjang badan, tinggi gumba dan lingkar dada pada umur sapih dan umur setahun sapi Bali di Balai Pembibitan Ternak Unggul (BPTU) Sapi Bali, Jembarana, Bali. Data yang digunakan adalah 120 catatan ukuran tubuh sapi Bali pada umur sapih dan umur setahun dari tahun 2006 sampai 2010. Data ukuran tubuh umur sapih dan umur setahun dianalisis untuk mendiskripsikan nilai rata-rata dan standar deviasi. Nilai heritabilitas diduga dengan menggunakan metode korelasi saudara tiri sebapak dengan metode Rancangan Acak Lengkap pola searah. Hasil estimasi tersebut menunjukkan bahwa rata-rata ukuran tubuh panjang badan, tinggi gumba dan lingkar dada pada umur sapih berturut-turut yaitu 94,70±4,42; 98,31±4,34 dan 120,78±7,39 cm serta pada umur setahun yaitu 95,90±4,46; 100,28±4,35 dan 125,45±7,40 cm. Hasil pendugaan nilai heritabilitas dengan menggunakan metode korelasi saudara tiri sebapak untuk ukuran tubuh panjang badan, tinggi gumba dan lingkar dada pada umur sapih berturut-turut adalah 0,92±0,0019; 0,76±0,0014 dan 0,56±0,0013 serta pada umur setahun yaitu 0,85±0,0015; 0,56±0,0010 dan 0,44±0,0009. Hasil dugaan nilai heritabilitas dapat disimpulkan bahwa nilai heritabilitas ukuran tubuh pada umur sapih dan umur setahun di Balai Pembibitan Ternak Unggul Sapi Bali, Jembrana, Bali bernilai tinggi.   (Kata kunci: Sapi Bali, Heritabilitas, Panjang badan, Tinggi gumba, Lingkar dada

PENDUGAAN NILAI HERITABILITAS UKURAN TUBUH PADA UMUR SAPIH DAN UMUR SETAHUN SAPI BALI DI BALAI PEMBIBITAN TERNAK UNGGUL SAPI BALI, JEMBRANA, BALI

Tujuan dari penelitian adalah untuk mengetahui nilai heritabilitas ukuran tubuh meliputi panjang badan, tinggi gumba dan lingkar dada pada umur sapih dan umur setahun sapi Bali di Balai Pembibitan Ternak Unggul (BPTU) Sapi Bali, Jembarana, Bali. Data yang digunakan adalah 120 catatan ukuran tubuh sapi Bali pada umur sapih dan umur setahun dari tahun 2006 sampai 2010. Data ukuran tubuh umur sapih dan umur setahun dianalisis untuk mendiskripsikan nilai rata-rata dan standar deviasi. Nilai heritabilitas diduga dengan menggunakan metode korelasi saudara tiri sebapak dengan metode Rancangan Acak Lengkap pola searah. Hasil estimasi tersebut menunjukkan bahwa rata-rata ukuran tubuh panjang badan, tinggi gumba dan lingkar dada pada umur sapih berturut-turut yaitu 94,70±4,42; 98,31±4,34 dan 120,78±7,39 cm serta pada umur setahun yaitu 95,90±4,46; 100,28±4,35 dan 125,45±7,40 cm. Hasil pendugaan nilai heritabilitas dengan menggunakan metode korelasi saudara tiri sebapak untuk ukuran tubuh panjang badan, tinggi gumba dan lingkar dada pada umur sapih berturut-turut adalah 0,92±0,0019; 0,76±0,0014 dan 0,56±0,0013 serta pada umur setahun yaitu 0,85±0,0015; 0,56±0,0010 dan 0,44±0,0009. Hasil dugaan nilai heritabilitas dapat disimpulkan bahwa nilai heritabilitas ukuran tubuh pada umur sapih dan umur setahun di Balai Pembibitan Ternak Unggul Sapi Bali, Jembrana, Bali bernilai tinggi. (Kata kunci: Sapi Bali, Heritabilitas, Panjang badan, Tinggi gumba, Lingkar dada

Variation of the breast cancer susceptibility marker, rs4245739, is associated with differential miRNA binding and MDM4 expression

A polymorphism, rs4245739, has been associated with susceptibility of several cancers including ER-negative breast cancer.  Rs4245739 is located at the 3’UTR of MDM4 gene, an oncogene that negatively regulates p53. The polymorphism has been associated with binding changes of miR-191. We studied, the influence of SNP rs4245739 to the binding of microRNAs, expression of microRNAs and MDM4. Using FindTar software, we detected potential microRNAs affected by the SNP-flanking sequence. We then used RNA sequencing data from ER-negative breast cancer to compare expression of miR-184, miR-191, miR-193a, miR-378, and MDM4 in different genotypes. Comparison of ER-negative patients with and without expression of miR-191 as well as profile microRNAs (miR-184, miR-191, miR-193a and miR-378 altogether) can differentiate expression of MDM4between different alleles. In addition, the number of lymphatic nodes affected in the individuals was also found to be significantly reduced in the risk group obtained by the miRNA profile method. We show our methods especially miRNA profile approach, are able to obtain new molecular and clinical features related to the rs4245739 SNP, a variant located in the 3’UTR of MDM4 gene and known to appear in different types of cancer. Keywords: ER negative breast cancer, rs4245739, microRNA, MDM4, p5

The accuracy of fine needle aspiration biopsy to diagnose breast neoplasm

Breast lump is a very common complaint among women, especially during the reproductive year. Fine needle aspiration biopsy (FNAB) is a less invasive procedure. It is usually performed as an initial diagnosis prior to the operative procedure. The accuracy of the FNAB in Indonesia needs to be elaborated. The study aimed to evaluate the sensitivity and specificity of FNAB in diagnosing breast neoplasm. This is a retrospective study with cross sectional design, involving 145 patients with breast lump who underwent FNAB and histopathology examination in Dr. Sardjito General Hospital, Yogyakarta, from 2012 to 2014. Data analysis showed that female to male ratio was 23. 2:1 commonly occurred at 41-50 years old. Forty-one cases (28.28%) diagnosed as a benign lesion with fibrocystic changes as the most frequentcase (11.19%). The malignant case was 104 cases (71.72%) with ductal carcinoma as the highest case (51.49%). FNAB achieved a sensitivity of 85.58%, a specificity of 100% and a total accuracy of 89.66% in determining the benign or malignant breast lump. The accuracy, sensitivity and specificity of FNAB in diagnosing ductal carcinoma were 83.58%, 85.51% and 81.54%, respectively. The accuracy, sensitivity and specificity of FNAB to diagnose fibrocystic changes lesion were 85.82%, 26.67% and 93.28%, respectively. FNAB can be used as an alternative diagnostic tool to diagnose breast neoplasm. It provides rapid, cheaper, effective, valuable, and less invasive procedure in diagnosis of breast lump.

Over- and down-expression mir-29c and mir-21 after chemotherapy and radio-therapy in nasopharyngeal carcinomas and the down-regulating proteins encoding eipstein barr virus and c-Myc.

Nasopharyngeal carcinoma (NPC) is the type of cancer related to multiple risk factors, including infection by Epstein Barr Virus (EBV). Standard treatment of NPC involves radiotherapy and chemotherapy in local and advanced tumors, while metastatic cases are treated with systemic chemotherapy. However, there is limited data on the causes of tumor recurrence, resistance, and progression. Moreover, the initial symptoms of NPC were often neglected until later enlarged, thus making it difficult to manage. MicroRNA (miRNA) is short molecule with 18-24 nucleotides and functions as protein-expression regulator protein in post-transcription. This study was aimed to determine miRNA expression and its relationship with the incidence of NPC. miR-21 and miR-29c were known to be involved in the development of NPC and resistance. A total of 51 plasma samples and 17 tissue samples were collected from Dharmais Hospital. The samples were taken from 17 untreated patients, 17 treated patients, and 17 healthy participants as control. We examined miRNA, protein of protein EBV (EBNA), and c-Myc expression using immunohistochemistry and quantitative polymerase chain reaction (qPCR). Our study revealed an increased expression of miR-21 and decreased expression of miR-29c in patients with NPC. There was also a correlation between the regulation of expression of miR-21 and c-Myc in the treated group of patients, and decreased expression in patients with complete response (CR) (4.13 ± 3.65: 2.74 ± 3.23; p <0.1). The parameters tend to increase in patients with partial response (PR) (3.00 ± 5, 86 compared to 8.77 ± 8.43; p <0.5), while no significant difference in expression of miR-29c in patients with CR and PR was detected. We concluded that miRNA might be detected in the plasma of NPC patients, and miR-21 might become a useful biomarker to determine therapeutic outcome in NPC patients.Keywords: nasopharyngeal cancer; miRNA; biomarke

The Expression of hsa-miR-155-5p in Plasma Samples Of Breast Cancer Before And After Chemotherapy

Breast cancer has emerged as the most common cancer-related mortality among women worldwide. Therefore, early cancer detection using biomarkers such as microRNA is needed. One of microRNAs that has an important role in breast cancer development is miR-155. Hsa-miR-155-5p is an oncomir that is commonly dysregulated in breast cancer. This study aims to determine the expression of hsa-miR-155-5p in breast cancer patient’s plasma before and after chemotherapy. We collected 64 samples from breast cancer patients admitted to Dr. Sardjito Hospital in Yogyakarta. RNA from plasma was extracted using RNA Isolation Kit miRCURY-Biofluid. cDNA synthesis was performed using cDNA Synthesis kit II and quantification of miR-155-5p using ExiLent SYBR Green master mix (Exiqon). qRT-PCR results were then analyzed with Livak's method and compared (before and after chemotherapy) with t-test. Expression of miR-155-5p in the breast cancer patients’ plasma after chemotherapy was significantly increased (10.59 times) when compared to before chemotherapy (p = 0.001). We concluded that there was upregulated expression of miR-155-5p after chemotherapy than before chemotherapy. There has not been a known, relevant pathway between hsa-miR-155-5p and chemotherapy regimens nor resistance to chemotherapy. Keywords: Breast cancer, plasma, hsa-miR-155-5p, oncomiR, chemotherapy

CURATION AND MANAGEMENT OF CULTURAL HERITAGE THROUGH LIBRARIES

Libraries, museums and archives hold valuable collections in a variety of media, presenting a vast body of knowledge rooted in the history of human civilisation. These form the repository of the wisdom of great works by thinkers of past and the present. The holdings of these institutions are priceless heritage of the mankind as they preserve documents, ideas, and the oral and written records. To value the cultural heritage and to care for it as a treasure bequeathed to us by our ancestors is the major responsibility of libraries. The past records constitute a natural resource and are indispensable to the present generation as well as to the generations to come. Libraries preserve the documentary heritage resources for which they are primarily responsible. Any loss of such materials is simply irreplaceable. Therefore, preserving this intellectual, cultural heritage becomes not only the academic commitment but also the moral responsibility of the librarians/information scientists, who are in charge of these repositories. The high quality of the papers and the discussion represent the thinking and experience of experts in their particular fields. The contributed papers also relate to the methodology used in libraries in Asia to provide access to manuscripts and cultural heritage. The volume discusses best practices in Knowledge preservation and how to collaborate and preserve the culture. The book also deals with manuscript and archives issues in the digital era. The approach of this book is concise, comprehensively, covering all major aspects of preservation and conservation through libraries. The readership of the book is not just limited to library and information science professionals, but also for those involved in conservation, preservation, restoration or other related disciplines. The book will be useful for librarians, archivists and conservators. We thank the Sunan Kalijaga University, Special Libraries Association- Asian Chapter for their trust and their constant support, all the contributors for their submissions, the members of the Local and International Committee for their reviewing effort for making this publication possible