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

Ali Rihani; Tom Van Maerken; Filip Pattyn; Gert Van Peer; Anneleen Beckers; Sara De Brouwer; Candy Kumps; Evelien Mets; Joni Van der Meulen; Pieter Rondou; Carina Leonelli; Pieter Mestdagh; Frank Speleman; Jo Vandesompele

doi:10.1371/journal.pone.0071776

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

PLoS One. 2013 Aug 14;8(8):e71776. doi: 10.1371/journal.pone.0071776. eCollection 2013.

Authors

Ali Rihani¹, Tom Van Maerken, Filip Pattyn, Gert Van Peer, Anneleen Beckers, Sara De Brouwer, Candy Kumps, Evelien Mets, Joni Van der Meulen, Pieter Rondou, Carina Leonelli, Pieter Mestdagh, Frank Speleman, Jo Vandesompele

Affiliation

¹ Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alu Elements*
Angiogenesis Inhibitors / pharmacology
Carrier Proteins / genetics
Carrier Proteins / metabolism
Cell Line, Tumor
Conserved Sequence
Gene Expression Profiling / standards*
Gene Knockdown Techniques
Humans
MicroRNAs / genetics
Protein Kinase Inhibitors / pharmacology
Pyrimidines / pharmacology
RNA, Small Interfering / genetics
Real-Time Polymerase Chain Reaction / standards*
Reference Standards
Repressor Proteins
Reverse Transcriptase Polymerase Chain Reaction / standards*
Transcriptome / drug effects
Tretinoin / pharmacology
Withanolides / pharmacology

Substances

Angiogenesis Inhibitors
Carrier Proteins
MicroRNAs
NVP-TAE684
PHF6 protein, human
Protein Kinase Inhibitors
Pyrimidines
RNA, Small Interfering
Repressor Proteins
Withanolides
Tretinoin
withaferin A

Grants and funding

Ali Rihani is supported by a PhD fellowship from the Ghent University research fund (BOF; 01D02210). Joni Van der Meulen and Evelien Mets are supported by a PhD fellowship from the Research Foundation – Flanders (FWO; 1116412N and 3G020209, respectively). Anneleen Beckers is supported by a PhD fellowship from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT; 101506). Tom Van Maerken, Pieter Mestdagh and Pieter Rondou are supported by postdoctoral fellowships from the FWO. SDB is supported by a postdoctoral fellowship from Ghent University (GOA UGENT; 01G01910). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.