Detectability of circulating microRNAs in microRNA extracts with Detectability of circulating microRNAs in microRNA extracts with low purity and yield using quantitative real-time polymerase chain low purity and yield using quantitative real-time polymerase chain reaction: Supporting evidence

Abstract

Detectability of circulating microRNAs in microRNA extracts with low purity and yield using quantitative real-time polymerase chain reaction: Supporting evidence

Azmir Ahmad 1 , Kahairi Abdullah , Mohd. Arifin Kaderi 2* , Afidalina Tumian 3 , Vijaya Mohan Sivanesan 4 5 , Muhammad Izani Mohd. Shiyuti , Wan Ishlah Leman 6 , Irfan Mohamad 7 , Wan Mohd. Nazri Wan Zainon 8 , Mark Paul 9 , Kamariah Mohamed Awang 9 , Luqman Rosla 10 10 , Sharifah Nor Ezura Syed Yussof

10 , Rosdi Ramli 11 1 Department of Basic Medical Science for Nursing, Kulliyyah of Nursing, International Islamic University Malaysia, Kuantan 25200, Malaysia 2 Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Kuantan 25200, Malaysia 3 PETRONAS Research Sdn. Bhd., Bandar Baru Bangi 43000, Malaysia 4 Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Shah Alam 40170, Malaysia 5 KPJ Batu Pahat Specialist Hospital, Batu Pahat 83000, Malaysia 6 Department of Otorhinolaryngology-Head & Neck Surgery, Kulliyyah of Medicine, International Islamic University Malaysia, Kuantan 25200, Malaysia 7 Department of Otorhinolaryngology-Head & Neck Surgery, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu 16150, Malaysia 8 Department of Nuclear Medicine, Radiotherapy and Oncology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu 16150, Malaysia 9 Department of Otorhinolaryngology, Hospital Tengku Ampuan Afzan, Kuantan 25100, Malaysia 10 Department of Otorhinolaryngology, Hospital Sultan Haji Ahmad Shah, Temerloh 28000, Malaysia 11 Department of Otorhinolaryngology, Hospital Raja Perempuan Zainab II, Kota Bharu 15200, Malaysia

* E-mail: ariffink@iium.edu.my

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.

Keywords: amplification, circulating, isolation and purification, microRNA, quantitative real-time polymerase chain reaction