Selection of suitable reference genes for quantitative real-time polymerase chain reaction in human meningiomas and arachnoidea
- Equal contributors
Department of Neurosurgery, University of Tuebingen, Hoppe-Seyler-Str.3, 72076 Tuebingen, Germany
BMC Research Notes 2011, 4:275 doi:10.1186/1756-0500-4-275Published: 2 August 2011
At first 32 housekeeping genes were analyzed in six randomly chosen meningiomas, brain and dura mater using geNorm, NormFinder, Bestkeeper-1 software and the comparative ΔCt method. Reference genes were ranked according to an integration tool for analyzing reference genes expression based on those four algorithms. Eight highest ranked reference genes (CASC3, EIF2B1, IPO8, MRPL19, PGK1, POP4, PPIA, and RPL37A) plus GAPDH and ACTB were then analyzed in 35 meningiomas, arachnoidea, dura mater and normal brain. NormFinder and Bestkeeper-1 identified RPL37A as the most stable expressed gene in meningiomas and their normal control tissue. NormFinder also determined the best combination of genes: RPL37A and EIF2B1. Commonly used reference genes GAPDH and ACTB were considered least stable genes. The critical influence of reference genes on qPCR data analysis is shown for VEGFA transcription patterns.
In meningiomas quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is most frequently used for accurate determination of gene expression using various reference genes. Although meningiomas are a heterogeneous group of tissue, no data have been reported to validate reference genes for meningiomas and their control tissues.
RPL37A is the optimal single reference gene for normalization of gene expression in meningiomas and their control tissues, although the use of the combination of RPL37A and EIF2B1 would provide more stable results.