- Department of Neurosurgery, School for Mental Health and Neuroscience, Maastricht University Medical Centre, Maastricht, The Netherlands
Department of Neurosurgery, School for Mental Health and Neuroscience, Maastricht University Medical Centre, Maastricht, The Netherlands
DOI:10.4103/sni.sni_399_16Copyright: © 2017 Surgical Neurology International This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
How to cite this article: R. G. C. Santegoeds, Y. Yakkioui, A. Jahanshahi, G. Hoogland, Y. Temel, J. J. van Overbeeke. Validation of reference genes in human chordoma. 05-Jun-2017;8:100
How to cite this URL: R. G. C. Santegoeds, Y. Yakkioui, A. Jahanshahi, G. Hoogland, Y. Temel, J. J. van Overbeeke. Validation of reference genes in human chordoma. 05-Jun-2017;8:100. Available from: http://surgicalneurologyint.com/surgicalint-articles/validation-of-reference-genes-in-human-chordoma/
Background:Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results.
Methods:The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes.
Results:From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results.
Conclusion:In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.
Keywords: Chordoma, GeNorn, NornFinder, qPCR, reference genes
Chordoma are rare tumors of the axial skeleton, which are thought to arise from remnants of the embryonic notochord.[
Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression. The greatest advantages of this technique are the sensitivity and accurate quantification.[
In chordoma, different reference genes have been used for normalization.[
Eighteen skull base chordoma tissue samples from 18 patients were collected in the course of resective therapy in the operation room. Upon collection, samples were flash frozen in liquid nitrogen immediately. A pathological diagnosis was made based on the World Health Organization classification of tumors. All tumors were diagnosed as a classical chordoma.
RNA isolation and cDNA synthesis
RNA was extracted from 30 mg of tissue with TRIzol® reagent (Invitrogen, Lot no. 10531501). After phase-separation with chloroform, the aqueous phase was mixed with 1 volume of isopropanol. The RNA was pelleted and washed twice with 75%, once with 100% ethanol, and dissolved in 30 μl DEPC treated water. RNA purity and yield were estimated by optical density using a NanoDrop ND-1000 spectrophotometer (Isogen Life Science). Trace amounts of DNA were removed by DNase I with a commercially available kit (#EN0521, Thermo Scientific) using 1 μg RNA and 1 U DNase I per reaction in a reaction volume of 11 μl, according to the manufacturer's protocol. DNase-treated RNA (1 μg per reaction) was then used as a template for cDNA synthesis using a RevertAid first strand cDNA synthesis kit (Thermo Scientific, #K1622, The Netherlands) containing oligo (dT)18 primers, according to the manufacturers’ protocol. A minus-reverse transcriptase (−RT) control was made for one sample to exclude genomic DNA contamination. All samples were diluted 1:5 and 1:50 in RNase free water, and then stored at −80°C until further analysis.
Twelve primers were designed to span an exon–intron boundary to exclude genomic DNA amplification. Reference genes were selected from different functional classes to avoid coregulation. The forward and reverse primer sequences are listed in
Reverse transcriptase quantitative PCR (RT-qPCR) was performed in a 96-well plate using a lightcycler 480 Real-Time PCR system (Roche applied science, Mannheim, Germany). The reaction conditions were set as follows: pre-incubation (5 min at 95°C), followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Finally, a melting curve was analyzed with a ramp rate of 0.29°C/s. The reaction volume was 20 μl, with 10 μl SYBR green mastermix (Roche Diagnostics GmbH, Mannheim, Germany, #13953820), 200 nM forward/reverse primers, and 2 μl of either 1:5 or 1:50 diluted cDNA, depending on gene expression. Per reference gene, all samples were measured in the same run in duplicate. Product specificity was confirmed by analyzing by the melting peak. Two negative controls were analyzed, one containing only the reaction reagents but no template and the –RT control.
RNA copy numbers were quantified using the comparative delta-delta-Ct method. The expression stability was tested with GeNorm[
Similarly, in NormFinder, genes with the lowest stability value have the most stable expression. However, unlike the geNorm algorithm, this algorithm does not calculate an optimal number of reference genes. The Microsoft Excel-based applet NormFinder (version 0.953) was used as described in a previous study.[
Twelve potential reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) were tested for expression stability on 18 flash frozen chordoma samples in duplicate. mRNA copy numbers were calculated using the delta–delta Ct method and the standard curve.[
Under these experimental conditions, geNorm analysis showed that PGK1, YWHAZ, and ACTB were the most stably expressed, whereas Brachyury was the least stable gene [
GeNorm determination of optimal number of reference genes, indicated by a GeNorm V value. This algorithm analyzes the stability when using multiple reference genes, and uses a cut-off value of 0.15 (indicated by a red line). When comparing the stability of 6 reference genes over 5 (V5/6), the value is 0.15, which means that there is no additional value of the usage of 6 reference genes. The most optimal number of reference genes is therefore 5
NormFinder analysis showed ACTB to be the most stably expressed reference gene, followed by YWHAZ, PGK1, B2M, and TBP [
In qRT-PCR, the analysis of constitutively expressed genes is often used to calibrate the expression level of the genes of interest. As the expression of genes can vary according to tissue type and experimental condition, the choice of reference genes may warrant a validation experiment. To the best of our knowledge, there is currently no data on the required set of reference genes to study gene expression in chordoma. In this study, we evaluated 12 candidate reference genes in flash frozen chordoma samples by using the frequently used algorithms geNorm and NormFinder.
Using the cut-off V-value 0.15, geNorm analysis indicated that the optimal set of reference genes consisted of PGK1, YWHAZ, ACTB, HPRT, and EF1a. In combination with the NormFinder data, the ultimate set of reference genes is ACTB, YWHAZ, PGK1, TBP, and HPRT. These results confirm the idea that chordoma are a very heterogeneous group of tumors since not just one or two but five reference genes are required.
Glyceralde-3-phosphate-dehydrogenase (GAPDH) has been frequently used as reference gene, including in many chordoma studies.[
In this study, we only used chordoma with brachyury expression. By immunohistochemistry, 81.1 to 100% of all chordoma are shown to be positive for brachyury.[
In this study, only tissue from skull based chordomas was used. Therefore, we cannot rule out possible differences in gene expression between cranial, sacral, or mobile spine chordoma. Another limitation of our study is that gene expression studies usually require control tissue. Since chordoma arises from remnants of the fetal notochord, this tissue can be considered to be the most suitable control. However, as notochord is difficult to dissect many studies have used nucleus pulposus tissue because it may still contain contain some notochordal cells.[
This is the first study that evaluated a panel of potential reference genes for chordoma. Using the geometric mean analysis of PGK1, ACTB, YWHAZ, TBP, and HPRT as reference genes allows a reliable interpretation of qRT-PCR data. The use of GAPDH as a single reference gene is not advised.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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