Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers

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 gene expression profiling. This approach is now being used in mammalian cell bioprocessing to...

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Main Authors:	Packeer Mohamed, Vasila, Hashim, Yumi Zuhanis Has-Yun, Amid, A., Mel, Maizirwan
Format:	Article
Language:	English
Published:	IIUM Press 2014
Subjects:	Q Science (General)
Online Access:	http://irep.iium.edu.my/38026/ http://irep.iium.edu.my/38026/ http://irep.iium.edu.my/38026/1/Paper_-_Packeer_Mohamed_2014_%28IIUM_EJ%29.pdf

id	iium-38026
recordtype	eprints
spelling	iium-380262014-10-22T10:41:49Z http://irep.iium.edu.my/38026/ Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers Packeer Mohamed, Vasila Hashim, Yumi Zuhanis Has-Yun Amid, A. Mel, Maizirwan Q Science (General) 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 gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help 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 was 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 μg/μl; p=0.004) than Total RNA purification kit (0.177 ± 0.0243 μg/μl). However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. 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 concern and Total RNA Purification kit (Norgen) is recommended if time and cost are concerned. IIUM Press 2014 Article PeerReviewed application/pdf en http://irep.iium.edu.my/38026/1/Paper_-_Packeer_Mohamed_2014_%28IIUM_EJ%29.pdf Packeer Mohamed, Vasila and Hashim, Yumi Zuhanis Has-Yun and Amid, A. and Mel, Maizirwan (2014) Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers. IIUM Engineering Journal, 15 (1). pp. 33-39. ISSN 1511-788X http://journals.iium.edu.my/ejournal/index.php/iiumej
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	Q Science (General)
spellingShingle	Q Science (General) Packeer Mohamed, Vasila Hashim, Yumi Zuhanis Has-Yun Amid, A. Mel, Maizirwan Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
description	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 gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help 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 was 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 μg/μl; p=0.004) than Total RNA purification kit (0.177 ± 0.0243 μg/μl). However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. 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 concern and Total RNA Purification kit (Norgen) is recommended if time and cost are concerned.
format	Article
author	Packeer Mohamed, Vasila Hashim, Yumi Zuhanis Has-Yun Amid, A. Mel, Maizirwan
author_facet	Packeer Mohamed, Vasila Hashim, Yumi Zuhanis Has-Yun Amid, A. Mel, Maizirwan
author_sort	Packeer Mohamed, Vasila
title	Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
title_short	Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
title_full	Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
title_fullStr	Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
title_full_unstemmed	Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers
title_sort	comparison of two total rna extraction protocols from cho-k1 cells for rt-pcr: cutoff cost for researchers
publisher	IIUM Press
publishDate	2014
url	http://irep.iium.edu.my/38026/ http://irep.iium.edu.my/38026/ http://irep.iium.edu.my/38026/1/Paper_-_Packeer_Mohamed_2014_%28IIUM_EJ%29.pdf
first_indexed	2023-09-18T20:54:34Z
last_indexed	2023-09-18T20:54:34Z
_version_	1777410217555787776

Comparison of two total RNA extraction protocols from CHO-K1 cells for RT-PCR: cutoff cost for researchers

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