Sains Malaysiana 47(2)(2018): 303-308

http://dx.doi.org/10.17576/jsm-2018-4702-12

 

Optimization of Illumina TruSeq Targeted RNA Expression (TREx) Library Quality

(Pengoptimuman Ekspresi RNA Sasaran (TREx) Illumina TruSeq Kualiti Perpustakaan)

 

NADIAH ABU^1,2, SWEE KEONG YEAP^3,4*, NORAINI NORDIN¹, SHEAU WEI TAN³,

WAN YONG HO⁵ & NOORJAHAN BANU ALITHEEN¹

 

¹Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

²UKM Molecular Biology Institute (UMBI), UKM Medical Centre, Jalan Yaa'cob Latiff

Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Wilayah Persekutuan, Malaysia

 

³Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor

Darul Ehsan, Malaysia

 

⁴China-ASEAN College of Marine Sciences, Xiamen University Malaysia, Jalan Sunsuria, Bandar Sunsuria, 43900 Sepang, Selangor Darul Ehsan, Malaysia

 

⁵School of Biomedical Sciences, The University of Nottingham Malaysia Campus,

Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 6 September 2016/Diterima: 2 Ogos 2017

 

ABSTRACT

 

RNA-seq has become an essential tool in molecular research. Nevertheless, application of RNA-seq was limited by cost and technical difficulties. Illumina has introduced the cost effective and ease to handle Truseq Targeted RNA Sequencing. In this study, we present the requirements and the optimization procedure for this Truseq Targeted RNA sequencing on cell line. Total RNA was recommended as starting materials but it required optimization including additional purification step and adjusting the AMPure beads ratio to eliminate unwanted contaminants. This can be resolved by using PolyA-enriched mRNA as starting material. TREx is a useful assay to evaluate gene expression. Quality library of TREx can be prepared by adding multiple washing steps or changing input sample to mRNA.

 

Keywords: Illumina; poly(A) mRNA; targeted expression; TREx

 

ABSTRAK

RNA-seq telah menjadi teknik penting di dalam kajian molekul. Walau bagaimanapun, kos yang tinggi serta kesukaran daripada segi teknikal telah menyebabkan kurangnya aplikasi RNA-seq. Illumina telah memperkenalkan teknik baru, penjujukan sasaran RNA Truseq yang lebih murah dan senang digunakan. Di dalam kajian ini, kami telah memperkenalkan syarat dan prosedur yang optimum bagi teknik ini. Penggunaan RNA keseluruhan sebagai bahan permulaan adalah digalakkan tetapi ia memerlukan kaedah pengoptimuman seperti pertambahan pembersihan dan penyesuaian nisbah manik ‘AMPure’ untuk menghapuskan bahan cemar yang tidak diingini. Ini boleh diselesaikan dengan menggunakan ‘PolyA-enriched’ mRNA sebagai bahan permulaan. TREx adalah asai berguna untuk menilai pengekspresan gen. Kualiti perpustakaan TREx boleh disediakan dengan menambah langkah basuhan berganda atau menukar sampel input kepada mRNA.

 

Kata kunci: Ekspresi sasaran; illumina; poli(A) mRNA; TREx

 

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*Pengarang untuk surat-menyurat; email: skyeap2005@gmail.com