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High-Throughput RT-qPCR for the Analysis of Circulating MicroRNAs

Tan GW, Tan LP

Methods Mol Biol, 2017;1580:7-19.
PMID: 28439823 DOI: 10.1007/978-1-4939-6866-4_2

Reverse transcription followed by real-time or quantitative polymerase chain reaction (RT-qPCR) is the gold standard for validation of results from transcriptomic profiling studies such as microarray and RNA sequencing. The current need for most studies, especially biomarker studies, is to evaluate the expression levels or fold changes of many transcripts in a large number of samples. With conventional low to medium throughput qPCR platforms, many qPCR plates would have to be run and a significant amount of RNA input per sample will be required to complete the experiments. This is particularly challenging when the size of study material (small biopsy, laser capture microdissected cells, biofluid, etc.), time, and resources are limited. A sensitive and high-throughput qPCR platform is therefore optimal for the evaluation of many transcripts in a large number of samples because the time needed to complete the entire experiment is shortened and the usage of lab consumables as well as RNA input per sample are low. Here, the methods of high-throughput RT-qPCR for the analysis of circulating microRNAs are described. Two distinctive qPCR chemistries (probe-based and intercalating dye-based) can be applied using the methods described here.
Fulltext Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

Tan GW, Khoo AS, Tan LP

Sci Rep, 2015;5:9430.
PMID: 25800946 DOI: 10.1038/srep09430

MicroRNAs regulate gene expression at the post-transcriptional level. Differential expression of miRNAs can potentially be used as biomarkers for early diagnosis and prediction for outcomes. Failure in validation of miRNA profiles is often caused by variations in experimental parameters. In this study, the performance of five extraction kits and three RT-qPCR systems were evaluated using BioMark high-throughput platform and the effects of different experimental parameters on circulating miRNA levels were determined. Differences in the performance of extraction kits as well as varying accuracy, sensitivity and reproducibility in qPCR systems were observed. Normalisation of RT-qPCR data to spike-in controls can reduce extraction bias. However, the extent of correlation for different qPCR systems varies in different assays. At different time points, there was no significant fold change in eight of the plasma miRNAs that we evaluated. Higher level of miRNAs was detected in plasma as compared to serum of the same cohort. In summary, we demonstrated that high-throughput RT-qPCR with pre-amplification step had increased sensitivity and can be achieved with accuracy and high reproducibility through stringent experimental controls. The information provided here is useful for planning biomarker validation studies involving circulating miRNAs.
Fulltext The disruptive mobile wallet in the hospitality industry: An extended mobile technology acceptance model

Lew S, Tan GW, Loh XM, Hew JJ, Ooi KB

Technol Soc, 2020 Nov;63:101430.
PMID: 33071395 DOI: 10.1016/j.techsoc.2020.101430

Given that the mobile wallet has become a disruptive innovation, especially in the Malaysian hospitality industry, this study investigates the adoption of mobile wallet in the hospitality industry among consumers. Different from the extant literature, this study extends the newly proposed mobile technology acceptance model with self-efficacy theory, critical mass theory, and flow theory to explain the behavioural intention to adopt. A self-administered questionnaire was utilised to collect data from mobile wallet users who have used a mobile wallet while dining out in restaurants and cafes. Other than technology self-efficacy and perceived critical mass, mobile usefulness, mobile ease of use, mobile self-efficacy, and perceived enjoyment were discovered to have a positive and significant association with the behavioural intention to adopt mobile wallet in the hospitality industry. The findings also suggested there were interrelationships between the constructs employed. Overall, the extended model was able to explain 61% of the variance in behavioural intention to adopt mobile wallet.
Fulltext The Microenvironment in Epstein-Barr Virus-Associated Malignancies

Tan GW, Visser L, Tan LP, van den Berg A, Diepstra A

Pathogens, 2018 04 13;7(2).
PMID: 29652813 DOI: 10.3390/pathogens7020040

The Epstein–Barr virus (EBV) can cause a wide variety of cancers upon infection of different cell types and induces a highly variable composition of the tumor microenvironment (TME). This TME consists of both innate and adaptive immune cells and is not merely an aspecific reaction to the tumor cells. In fact, latent EBV-infected tumor cells utilize several specific mechanisms to form and shape the TME to their own benefit. These mechanisms have been studied largely in the context of EBV+ Hodgkin lymphoma, undifferentiated nasopharyngeal carcinoma, and EBV+ gastric cancer. This review describes the composition, immune escape mechanisms, and tumor cell promoting properties of the TME in these three malignancies. Mechanisms of susceptibility which regularly involve genes related to immune system function are also discussed, as only a small proportion of EBV-infected individuals develops an EBV-associated malignancy.
Fulltext The Dark Side of Mobile Learning via Social Media: How Bad Can It Get?

Loh XK, Lee VH, Loh XM, Tan GW, Ooi KB, Dwivedi YK

Inf Syst Front, 2022;24(6):1887-1904.
PMID: 34658660 DOI: 10.1007/s10796-021-10202-z

As the COVID-19 pandemic continues to spread at an unprecedented rate, many universities around the world halted physical forms of teaching and learning to stop the spread of the virus. As a result, many university students were forced to utilize online learning through channels such as mobile social media. Due to the novelty of this situation, there are many unknowns particularly with the negative influences of mobile learning via social media on university students. Thus, this study looks to examine this subject matter from the perspective of the stimulus-organism-response theory. The uniquely developed research model included four stimuli (i.e., social overload, information overload, life invasion, and privacy invasion), two organisms (i.e., technostress and exhaustion) as well as a response in terms of reduced intention to use mobile learning via social media. The responses were collected from 384 university students via an online survey and analyzed with the Partial-Least-Square-Structural-Equation-Modelling. It was found that the antecedents for both technostress and exhaustion were able to account for more than half of their respective variances. Furthermore, technostress and exhaustion were significant facilitators of the students' reduced intention to use mobile learning via social media. In addition to the practical insights for stakeholders in the education industry, this study also posited several theoretical implications for researchers.
Fulltext The survey dataset of The Influence of theory of planned behaviour on purchase behaviour on social media

Zhou Y, Loi AM, Tan GW, Lo PS, Lim W

Data Brief, 2022 Jun;42:108239.
PMID: 35592771 DOI: 10.1016/j.dib.2022.108239

The research aims to study the correlations between attitudes of users, social norms, perceived behavioural control, and purchase behaviours. The research population consists of social media users in Malaysia. The data was collected from 205 respondents via a self-administered online survey. The theory of planned behaviour acts as the underlying theory in the research. Next, descriptive and hypothesis-testing quantitative analysis were adopted to probe the relationships between the variables. Moreover, G*Power was used to identify the minimum sample size, and SPSS v.22 was employed to examine the datasets.
Fulltext HLA Expression in Relation to HLA Type in Classic Hodgkin Lymphoma Patients

Tan GW, Jiang P, Nolte IM, Kushekhar K, Veenstra RN, Hepkema BG, et al.

Cancers (Basel), 2021 Nov 20;13(22).
PMID: 34830986 DOI: 10.3390/cancers13225833

Several human leukocyte antigen (HLA) alleles are strongly associated with susceptibility to classic Hodgkin lymphoma (cHL), also in subgroups stratified for presence of the Epstein-Barr virus (EBV). We tested the hypothesis that the pressure on cHL tumour cells to lose HLA expression is associated with HLA susceptibility alleles. A meta-analysis was carried out to identify consistent protective and risk HLA alleles in a combined cohort of 839 cHL patients from the Netherlands and the United Kingdom. Tumour cell HLA expression was studied in 338 cHL cases from these two cohorts and correlated to the presence of specific susceptibility HLA alleles. Carriers of the HLA-DRB1*07 protective allele frequently lost HLA class II expression in cHL overall. Patients carrying the HLA-DRB1*15/16 (DR2) risk allele retained HLA class II expression in EBV- cHL and patients with the HLA-B*37 risk allele retained HLA class I expression more frequently than non-carriers in EBV+ cHL. The other susceptibility alleles showed no significant differences in expression. Thus, HLA expression by tumour cells is associated with a subset of the protective and risk alleles. This strongly suggests that HLA associations in cHL are related to peptide binding capacities of specific HLA alleles.
Fulltext A novel and non-invasive approach utilising nasal washings for the detection of nasopharyngeal carcinoma

Tan GW, Sivanesan VM, Abdul Rahman FI, Hassan F, Hasbullah HH, Ng CC, et al.

Int J Cancer, 2019 Oct 15;145(8):2260-2266.
PMID: 30698824 DOI: 10.1002/ijc.32173

Nasopharyngeal carcinoma (NPC) is an epithelial cancer of the nasopharynx which is highly associated with Epstein-Barr virus (EBV). Worldwide, most of the top 20 countries with the highest incidence and mortality rates of NPC are low- and middle-income countries. Many studies had demonstrated that EBV could be detected in the tissue, serum and plasma of NPC patients. In this study, we explored the potential of assays based on non-invasive nasal washings (NW) as a diagnostic and prognostic tool for NPC. A total of 128 patients were evaluated for NW EBV DNA loads and a subset of these samples were also tested for 27 EBV and human miRNAs shortlisted from literature. EBV DNA and seven miRNAs showed area under the receiver operating characteristic curve (AUC) values of more than 0.7, suggestive of their potential utility to detect NPC. Logistic regression analyses suggested that combination of two NW assays that test for EBNA-1 and hsa-miR-21 had the best performance in detecting NPC. The trend of NW EBV DNA load matched with clinical outcome of 71.4% (10 out of 14) NPC patients being followed-up. In summary, the non-invasive NW testing panel may be particularly useful for NPC screening in remote areas where healthcare facilities and otolaryngologists are lacking, and may encourage frequent testing of individuals in the high risk groups who are reluctant to have their blood tested. However, further validation in an independent cohort is required to strengthen the utility of this testing panel as a non-invasive detection tool for NPC.
Fulltext Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR

Zhong Y, Tan GW, Bult J, Veltmaat N, Plattel W, Kluiver J, et al.

BMC Cancer, 2024 Apr 02;24(1):407.
PMID: 38566053 DOI: 10.1186/s12885-024-12191-z

BACKGROUND: Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL.
METHODS: We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively.
RESULTS: MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196.
CONCLUSION: The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.
Fulltext Integrated pathway analysis of nasopharyngeal carcinoma implicates the axonemal dynein complex in the Malaysian cohort

Chin YM, Tan LP, Abdul Aziz N, Mushiroda T, Kubo M, Mohd Kornain NK, et al.

Int J Cancer, 2016 10 15;139(8):1731-9.
PMID: 27236004 DOI: 10.1002/ijc.30207

Nasopharyngeal carcinoma (NPC) is an epithelial squamous cell carcinoma on the mucosal lining of the nasopharynx. The etiology of NPC remains elusive despite many reported studies. Most studies employ a single platform approach, neglecting the cumulative influence of both the genome and transcriptome toward NPC development. We aim to employ an integrated pathway approach to identify dysregulated pathways linked to NPC. Our approach combines imputation NPC GWAS data from a Malaysian cohort as well as published expression data GSE12452 from both NPC and non-NPC nasopharynx tissues. Pathway association for GWAS data was performed using MAGENTA while for expression data, GSA-SNP was used with gene p values derived from differential expression values from GEO2R. Our study identified NPC association in the gene ontology (GO) axonemal dynein complex pathway (pGWAS-GSEA = 1.98 × 10(-2) ; pExpr-GSEA = 1.27 × 10(-24) ; pBonf-Combined = 4.15 × 10(-21) ). This association was replicated in a separate cohort using gene expression data from NPC and non-NPC nasopharynx tissues (pAmpliSeq-GSEA = 6.56 × 10(-4) ). Loss of function in the axonemal dynein complex causes impaired cilia function, leading to poor mucociliary clearance and subsequently upper or lower respiratory tract infection, the former of which includes the nasopharynx. Our approach illustrates the potential use of integrated pathway analysis in detecting gene sets involved in the development of NPC in the Malaysian cohort.
Fulltext Systematic comparison of plasma EBV DNA, anti-EBV antibodies and miRNA levels for early detection and prognosis of nasopharyngeal carcinoma

Tan LP, Tan GW, Sivanesan VM, Goh SL, Ng XJ, Lim CS, et al.

Int J Cancer, 2020 04 15;146(8):2336-2347.
PMID: 31469434 DOI: 10.1002/ijc.32656

Nasopharyngeal carcinoma (NPC) is originated from the epithelial cells of nasopharynx, Epstein-Barr virus (EBV)-associated and has the highest incidence and mortality rates in Southeast Asia. Late presentation is a common issue and early detection could be the key to reduce the disease burden. Sensitivity of plasma EBV DNA, an established NPC biomarker, for Stage I NPC is controversial. Most newly reported NPC biomarkers have neither been externally validated nor compared to the established ones. This causes difficulty in planning for cost-effective early detection strategies. Our study systematically evaluated six established and four new biomarkers in NPC cases, population controls and hospital controls. We showed that BamHI-W 76 bp remains the most sensitive plasma biomarker, with 96.7% (29/30), 96.7% (58/60) and 97.4% (226/232) sensitivity to detect Stage I, early stage and all NPC, respectively. Its specificity was 94.2% (113/120) against population controls and 90.4% (113/125) against hospital controls. Diagnostic accuracy of BamHI-W 121 bp and ebv-miR-BART7-3p were validated. Hsa-miR-29a-3p and hsa-miR-103a-3p were not, possibly due to lower number of advanced stage NPC cases included in this subset. Decision tree modeling suggested that combination of BamHI-W 76 bp and VCA IgA or EA IgG may increase the specificity or sensitivity to detect NPC. EBNA1 99 bp could identify NPC patients with poor prognosis in early and advanced stage NPC. Our findings provided evidence for improvement in NPC screening strategies, covering considerations of opportunistic screening, combining biomarkers to increase sensitivity or specificity and testing biomarkers from single sampled specimen to avoid logistic problems of resampling.

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