Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cut-off cost for researchers. IIUM Engineering Journal, 15 (1). pp. 33-39. ISSN 1511-788X (2014)
Abstract
Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR) is a valuable tool used to study the gene expression profiling. This approach is now being used in mammalian cell bioprocessing for helping to understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples were involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada) and RNeasy mini kit (with DNase treatment; Qiagen, USA) respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 μ g/μl; p=0.004) than Total RNA purification kit (0.177 ± 0.0243 μ g/μ l). However, the RNA purity of both methods was close to 2.0 and there was no significant difference between the methods. The total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp. Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen) is recommended if higher yields is the primary issue and the Total RNA Purification kit (Norgen) is recommended if time and cost are concerned.
| Item Type: | Article |
|---|---|
| Keywords: | CHO-K1, RNA extraction, Reverse transcription polymerase chain reaction (RT-PCR) |
| Taxonomy: | By Subject > College of Engineering > Chemical Engineering > Biotechnology & Bioprocess |
| Local Content Hub: | Subjects > College of Engineering |
| Depositing User: | Eza Eliana Abdul Wahid |
| Date Deposited: | 30 Dec 2022 08:50 |
| Last Modified: | 16 Jul 2024 00:02 |
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