ACTB and SDHA Are Suitable Endogenous Reference Genes for Gene Expression Studies in Human Astrocytomas Using Quantitative RT-PCR

Abstract

Background: Quantitative real-time reverse-transcription polymerase chain reaction is frequently used as research tool in experimental oncology. There are some studies of valid endogenous control genes in the field of human glioma research, which, however, only focus on the comparison between normal brain with tumor tissue and malignant transformation toward secondary glioblastomas. Aim of this study was to validate a more general reference gene also suitable for pre- and posttreatment analysis and other evaluations (eg, primary vs secondary glioblastoma).

Methods: This quantitative polymerase chain reaction analysis was performed to test a panel of the 6 most suitable reference genes from other studies representing different physiological pathways (ACTB, GAPDH, POLR2A, RPL13A, SDHA, and TBP) in all common glioma groups, namely: diffuse astrocytoma World Health Organization II, anaplastic astrocytoma World Health Organization III, secondary glioblastoma World Health Organization IV with and without chemotherapy, primary glioblastoma, recurrent glioblastoma, and gliomas before and after radiation. Expression stability was tested during the longitudinal course of the disease in 8 single patients.

Results: Evaluation of the expression levels of the 6 target genes showed that ACTB, GAPDH, and RPL13A show higher expression compared to SDHA, POLR2A, and TBP. ACTB, GAPDH, and RPL13A showed different expression levels between astrozytoma grade II and primary glioblastoma. Except for this difference, the candidate genes were not differentially expressed between primary and secondary glioblastomas and between the World Health Organization tumor grades. Furthermore, they remained stable before and after radiotherapy and/or chemotherapy. Therefore, they are adequate references for glioblastoma gene expression studies. The comparison of all tested genes resulted in SDHA and ACTB as most stable reference genes determined by the NormFinder software. Our data revealed lowest intragroup variation in the SDHA, highest in the RPL13A gene.

Conclusions: All tested genes may be recommended as universal reference genes for data normalization in gene expression studies under different treatment regimens both in primary glioblastomas and astrocytomas of different grades (World Health Organization grades II-IV), respectively. In summary, ACTB and SDHA exhibited the best stability values and showed the lowest intergroup expression variability.

Keywords: WHO grade; astrocytoma; glioblastoma multiforme; glioma; housekeeping gene; qPCR; quantitative RT-PCR; quantitative real-time polymerase chain reaction; reference gene.

Figure 1.

Comparison of threshold cycles (Ct values). GIIIR indicates anaplastic astrocytome grade III; pGBM, primary glioblastoma; pGBM R + C, primary recurrent glioblastoma with chemotherapy; pGII, astrocytoma grade II; sGBM R + C, secondary glioblastoma with chemotherapy; sGBM R − C, secondary glioblastoma without chemotherapy.

Figure 2.

Comparison of World Health Organization (WHO) II, III, and IV Gliomas: Ct values of astrocytoma grade II (white), grade III (gray), and secondary glioblastoma (GBM; black).

Figure 3.

Primary glioblastoma (GBM) compared to secondary GBM (black).

Figure 4.

Primary glioblastoma (GBM; black) versus recurrent primary GBM (+CTx).

Figure 5.

Secondary glioblastoma (GBM) with/without chemotherapy.

Figure 6.

Gliomas with/without radiotherapy.

Figure 7.

Intergroup variation. GIIIR indicates anaplastic astrocytome grade III; pGBM, primary glioblastoma; pGBM R + C, primary recurrent glioblastoma with chemotherapy; pGII, astrocytoma grade II; sGBM R + C, secondary glioblastoma with chemotherapy; sGBM R − C, secondary glioblastoma without chemotherapy.

Figure 8.

Longitudinal course with malignant transformation in 3 single patients. Expression of candidate reference genes (Ct values) in 3 patients during progress of recurrency, ♦ glioma II, ▴ glioma III, ▪ glioblastoma.