gms | German Medical Science

Objective: Real-time RT-PCR (RT-qPCR) is frequently used to analyse gene expression. In human astrocytomas, three studies have been performed validating reference genes, however, no systematic analysis of suitable endogenous control genes exists for recurrent tumors and under treatment conditions. Therefore, this analysis enables longitudinally designed studies including pre- and post-chemotherapy, after radiotherapy and in astrocytomas before and after malignant transformation.

Method: This study aimed to test the best six reference genes out of the earlier studies (RPL13A, GAPDH, SDHA, POLR2A, ACTB, and TBP) for their expression stability in the following groups: (i) diffuse astrocytoma, anaplastic astrocytoma, and glioblastoma (GBM); (ii) primary and secondary GBM; (iii) primary and recurrent glioma; (iv) with and without chemotherapy (CTx), and (v) before and after radiotherapy. Tissue samples (n=10 per group) were obtained from our tumour tissue bank. After RNA extraction and transcription into cDNA, all qPCRs were done twice in duplicate.

Results: The CT-values of the reference genes analyzed showed different transcription levels in all groups, ranging from 14–16 (ACTB, GAPDH, RPL13A, high expression) to 20–23 (SDHA, POLR2A, TBP, low expression). The candidate genes were not differentially expressed between primary and secondary glioblastomas. Furthermore, they remained stable before and after radiotherapy and / or chemotherapy. Only ACTB, GAPDH and RPL13A showed significantly different expression levels between astrocytoma grade II and primary glioblastoma. However, these groups are unlikely to be compared. Therefore, they are adequate references for GBM gene expression studies. Moreover, the comparison betweem different glioma entities resulted in SDHA and ACTB being the most stable reference genes determined by the NormFinder software. The data revealed lowest intragroup variation in the SDHA, highest in the RPL13A gene. Our results indicate that SDHA displays the most stable values for recurrent astrocytomas with rising tumor grade (WHO grade ll to III to IV). ACTB shows the most stable results as a gene which is highly expressed in brain tumor tissue.

Conclusions: In summary, our data show the relevance of previous validation of candidate control genes for each experimental design and indicates that the most suitable housekeeping genes are SDHA and ACTB for quantitative PCR analysis in glioma tissue of different malignancies, after recurrence, and under different treatment conditions.