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Effective Alu repeat based RT-qPCR normalization in cancer cell perturbation experiments

Ali Rihani (UGent) , Tom Van Maerken (UGent) , Filip Pattyn, Gert Van Peer (UGent) , Anneleen Beckers (UGent) , Sara De Brouwer (UGent) , Candy Kumps (UGent) , Evelien Mets (UGent) , Joni Van der Meulen (UGent) , Pieter Rondou (UGent) , et al.
(2013) PLOS ONE. 8(8).
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Abstract
Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. Results: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5). Conclusions: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.
Keywords
GENE-EXPRESSION, REAL-TIME PCR, NEUROBLASTOMA-CELLS, RIBOSOMAL-RNA, INAPPROPRIATE, INHIBITOR, MESSENGER, TISSUE, TUMORS

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Chicago
Rihani, Ali, Tom Van Maerken, Filip Pattyn, Gert Van Peer, Anneleen Beckers, Sara De Brouwer, Candy Kumps, et al. 2013. “Effective Alu Repeat Based RT-qPCR Normalization in Cancer Cell Perturbation Experiments.” Plos One 8 (8).
APA
Rihani, A., Van Maerken, T., Pattyn, F., Van Peer, G., Beckers, A., De Brouwer, S., Kumps, C., et al. (2013). Effective Alu repeat based RT-qPCR normalization in cancer cell perturbation experiments. PLOS ONE, 8(8).
Vancouver
1.
Rihani A, Van Maerken T, Pattyn F, Van Peer G, Beckers A, De Brouwer S, et al. Effective Alu repeat based RT-qPCR normalization in cancer cell perturbation experiments. PLOS ONE. 2013;8(8).
MLA
Rihani, Ali, Tom Van Maerken, Filip Pattyn, et al. “Effective Alu Repeat Based RT-qPCR Normalization in Cancer Cell Perturbation Experiments.” PLOS ONE 8.8 (2013): n. pag. Print.
@article{4117514,
  abstract     = {Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations.
Results: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M{\textlangle}0.5).
Conclusions: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.},
  articleno    = {e71776},
  author       = {Rihani, Ali and Van Maerken, Tom and Pattyn, Filip and Van Peer, Gert and Beckers, Anneleen and De Brouwer, Sara and Kumps, Candy and Mets, Evelien and Van der Meulen, Joni and Rondou, Pieter and Leonelli, Carina and Mestdagh, Pieter and Speleman, Franki and Vandesompele, Jo},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  language     = {eng},
  number       = {8},
  pages        = {8},
  title        = {Effective Alu repeat based RT-qPCR normalization in cancer cell perturbation experiments},
  url          = {http://dx.doi.org/10.1371/journal.pone.0071776},
  volume       = {8},
  year         = {2013},
}

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