COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS
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 gene expression profiling. This approach is now being used in mammalian cell bioproc...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
IIUM Press, International Islamic University Malaysia
2014-05-01
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| Series: | International Islamic University Malaysia Engineering Journal |
| Online Access: | http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/452 |
| _version_ | 1811276560355819520 |
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| author | Vasila Packeer Mohamed Yumi Z. H-Y. Hashim A. Amid M. Mel |
| author_facet | Vasila Packeer Mohamed Yumi Z. H-Y. Hashim A. Amid M. Mel |
| author_sort | Vasila Packeer Mohamed |
| collection | DOAJ |
| description | 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 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.
ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat. Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR) merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil. Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem. Tujuan kajian dijalankan adalah untuk menentukan dan membandingkan kaedah ekstraksi RNA yang paling sesuai bagi sel CHO-K1 di persekitaran di mana kadar sampel yang agak besar terlibat. Jumlah RNA diekstrak menggunakan kit penulenan Jumlah RNA (tanpa rawatan DNase; Norgen, Canada) dan kit mini RNeasy (dengan rawatan DNase; Qiagen, USA). RNA yang diekstrak kemudiannya diterbalikkan transkripsi, dan cDNA menjalani penguat PCR 18S. Hasil daripada kit RNeasy adalah lebih tinggi (0.316 ± 0.033 µg/µl; p=0.004) berbanding dengan kit penulenan Jumlah RNA (0.177 ± 0.0243 µg/µl). Walaupun begitu, kaedah penulenan RNA untuk kedua-duanya hampir 2.0 dan tidak terdapat perbezaan yang ketara antara keduanya. Kit penulenan Jumlah RNA adalah lebih murah berbanding dengan kit RNeasy. Memandangkan tidak ada langkah rawatan DNase dengan penggunaan kit Jumlah RNA, tempoh ekstrak RNA nya lebih pendek. Apabila RNA yang telah diekstrak menjalani RT-PCR, kedua-dua kaedah berjaya mengesan 18S pada 219 bp. Kesimpulannya, kajian ini menunjukkan kedua-dua kaedah sesuai untuk mengekstrak RNA bagi sel CHO-K1. Kit mini RNeasy (Qiagen) lebih sesuai jika hasil yang tinggi diinginkan dan kit penulenan Jumlah RNA (Norgen) pula ideal, jika kos dan masa berkepentingan. |
| first_indexed | 2024-04-12T23:58:45Z |
| format | Article |
| id | doaj.art-113bc62c67834eb69d4df6dc45d7406a |
| institution | Directory Open Access Journal |
| issn | 1511-788X 2289-7860 |
| language | English |
| last_indexed | 2024-04-12T23:58:45Z |
| publishDate | 2014-05-01 |
| publisher | IIUM Press, International Islamic University Malaysia |
| record_format | Article |
| series | International Islamic University Malaysia Engineering Journal |
| spelling | doaj.art-113bc62c67834eb69d4df6dc45d7406a2022-12-22T03:11:24ZengIIUM Press, International Islamic University MalaysiaInternational Islamic University Malaysia Engineering Journal1511-788X2289-78602014-05-0115110.31436/iiumej.v15i1.452COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERSVasila Packeer Mohamed0Yumi Z. H-Y. Hashim1A. Amid2M. Mel3Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia (IIUM), P.O. Box 10, 50728, Kuala Lumpur, Malaysia.Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia (IIUM), P.O. Box 10, 50728, Kuala Lumpur, Malaysia.Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia (IIUM), P.O. Box 10, 50728, Kuala Lumpur, Malaysia.Bioprocess and Molecular Engineering Research Unit (BPMERU), Department of Biotechnology Engineering, Kulliyyah of Engineering, International Islamic University Malaysia (IIUM), P.O. Box 10, 50728, Kuala Lumpur, Malaysia.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 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. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat. Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR) merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil. Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem. Tujuan kajian dijalankan adalah untuk menentukan dan membandingkan kaedah ekstraksi RNA yang paling sesuai bagi sel CHO-K1 di persekitaran di mana kadar sampel yang agak besar terlibat. Jumlah RNA diekstrak menggunakan kit penulenan Jumlah RNA (tanpa rawatan DNase; Norgen, Canada) dan kit mini RNeasy (dengan rawatan DNase; Qiagen, USA). RNA yang diekstrak kemudiannya diterbalikkan transkripsi, dan cDNA menjalani penguat PCR 18S. Hasil daripada kit RNeasy adalah lebih tinggi (0.316 ± 0.033 µg/µl; p=0.004) berbanding dengan kit penulenan Jumlah RNA (0.177 ± 0.0243 µg/µl). Walaupun begitu, kaedah penulenan RNA untuk kedua-duanya hampir 2.0 dan tidak terdapat perbezaan yang ketara antara keduanya. Kit penulenan Jumlah RNA adalah lebih murah berbanding dengan kit RNeasy. Memandangkan tidak ada langkah rawatan DNase dengan penggunaan kit Jumlah RNA, tempoh ekstrak RNA nya lebih pendek. Apabila RNA yang telah diekstrak menjalani RT-PCR, kedua-dua kaedah berjaya mengesan 18S pada 219 bp. Kesimpulannya, kajian ini menunjukkan kedua-dua kaedah sesuai untuk mengekstrak RNA bagi sel CHO-K1. Kit mini RNeasy (Qiagen) lebih sesuai jika hasil yang tinggi diinginkan dan kit penulenan Jumlah RNA (Norgen) pula ideal, jika kos dan masa berkepentingan.http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/452 |
| spellingShingle | Vasila Packeer Mohamed Yumi Z. H-Y. Hashim A. Amid M. Mel COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS International Islamic University Malaysia Engineering Journal |
| title | COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS |
| title_full | COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS |
| title_fullStr | COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS |
| title_full_unstemmed | COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS |
| title_short | COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS |
| title_sort | comparison of two total rna extraction protocols from cho k1 cells for rt pcr cut off cost for researchers |
| url | http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/452 |
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