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All Journal HAYATI Journal of Biosciences Makara Journal of Health Research
Syed Yussof, Sharifah Nor Ezura
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Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Detectability of circulating microRNAs in microRNA extracts with low purity and yield using quantitative real-time polymerase chain reaction: Supporting evidence Ahmad, Azmir; Kaderi, Mohd. Arifin; Tumian, Afidalina; Sivanesan, Vijaya Mohan; Abdullah, Kahairi; Leman, Wan Ishlah; Mohamad, Irfan; Wan Zainon, Wan Mohd. Nazri; Mohd. Shiyuti, Muhammad Izani; Mohamed Awang, Kamariah; Rosla, Luqman; Paul, Mark; Syed Yussof, Sharifah Nor Ezura; Ramli, Rosdi
Makara Journal of Health Research Vol. 24, No. 3
Publisher : UI Scholars Hub

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Abstract

Background: Circulating microRNAs (miRNAs) are a group of noncoding RNAs with promising potential as minimal invasive biomarkers for noncommunicable diseases. However, challenges exist in the preparation of these miRNAs from peripheral blood samples for quantification purposes. The low quality of miRNA extracts presents an obstacle. Acknowledging the superior performance of quantitative real-time polymerase chain reaction (qPCR) as gold standard for gene expression analysis, we conducted this study to observe the capabilities of qPCR using the Taqman® protocol in amplifying circulating miRNAs from miRNA extracts with low purity and yield. Methods: miRNAs were extracted from thirty-six plasma samples that were obtained from public subjects. Four selected miRNAs were quantified using the Taqman® protocol in an integrated fluidic circuit chip that was optimized from a previous study. The amplification graph and Cq values were obtained to observe any abnormal amplification signs and expression levels, respectively. Results: The qualitative observation of the amplification of the four miRNAs showed no sign of abnormality, thereby indicating the successful amplification of the miRNAs without enzymatic inhibition. Furthermore, the miRNAs were quantified in high expression levels. Conclusion: The circulating miRNA extracts with low purity and yield were practical for the study of circulating miRNA expression based on the Taqman® protocol as the method of detection.
Quantile Normalization for High Throughput Circulating MicroRNA Expression Study using TaqMan® Low Density Array Panels: Supporting Evidence Ahmad, Azmir; Mohamed, Syarah Syamimi; Tumian, Afidalina; Tolos, Siti Marponga; Sivanesan, Vijaya Mohan; Leman, Wan Ishlah; Abdullah, Kahairi; Mohamad, Irfan; Wan Zainon, Wan Mohd. Nazri; Rosla, Luqman; Syed Yussof, Sharifah Nor Ezura; Mark, Paul; Mohamed@Awang, Kamariah; Ramli, Rosdi; Omar, Eshamsol Kamar; Mohd. Yassin, Mohd. Wardah; Mohamad, Mohd. Amin Marwan; Kaderi, Mohd. Arifin
HAYATI Journal of Biosciences Vol. 31 No. 3 (2024): May 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.3.432-442

Abstract

In searching for new biomarkers, high throughput technique has been widely used by researchers, including for gene expression study. However, the reliability and accuracy of results from high throughput study critically depends on appropriate data management, including normalization methods. Data driven normalization has been introduced as a normalization method for high throughput gene expression study. Thus, this study was conducted to evaluate the performance of various data driven and reference genes normalization methods using a high throughput circulating microRNA expression dataset. A quantification cycle (Cq) dataset generated from a high throughput circulating microRNA study was used to test the normalization methods using HTqPCR package in R software. The normalized Cq generated from different methods were compared descriptively using box plot analysis and coefficient of variance. The box plot analysis showed that quantile normalization produced more homogenous Cq distribution, lesser outliers and reduced coefficient of variance as compared to other normalization methods in screening and validation phases. The overview on quantile normalized Cq showed consistency in its level of expression before and after 2-∆∆Cq calculation indicating the reliability of quantile normalized Cq. Quantile normalization is suggested to be used in high throughput miRNA expression study due to its performance in homogenizing the data, reduce outliers and coefficient of variance.
Co-Authors Abdullah, Kahairi Ahmad, Azmir Kaderi, Mohd. Arifin Leman, Wan Ishlah Mark, Paul Mohamad, Irfan Mohamad, Mohd. Amin Marwan Mohamed Awang, Kamariah Mohamed, Syarah Syamimi Mohamed@Awang, Kamariah Mohd. Shiyuti, Muhammad Izani Mohd. Yassin, Mohd. Wardah Omar, Eshamsol Kamar Paul, Mark Ramli, Rosdi Rosla, Luqman Sivanesan, Vijaya Mohan Tolos, Siti Marponga Tumian, Afidalina Wan Zainon, Wan Mohd. Nazri