Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue

Abstract

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate dehydrogenase complex, subunit A, cyclophilin A and ubiquitin C. The aim of the current study was to assess which reference genes show stable mRNA levels in human post mortem cardiac muscle, skeletal muscle and brain tissue. Considering cardiac muscle tissue, CYCA and TBP were identified as the most stable while in skeletal muscle tissue, SDHA and TBP, and in brain tissue, SDHA and HMBS turned out to be the most stable. Furthermore, we recommend a minimum of four carefully validated endogenous control genes for reliable data normalisation in human post mortem tissue. Parameters influencing the stability of transcript amounts were found to be mainly the post mortem interval in cardiac muscle and skeletal muscle tissue and the donor's cause of death in skeletal muscle and brain samples. Further parameters like gender, age at death and body mass index were found to influence mRNA quantities in skeletal muscle only. The set of stable control genes identified in this study may be used in further studies if the composition of the samples is similar to the one used here.

RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays

“…Thus, even though the RIN reveals the integrity of total RNA and the messenger RNA (mRNA) fraction is only approximately 3% of total RNA, the RIN was found to be a very good indicator for mRNA integrity, too. The highest observed shift was 2.60 (18S rRNA in heart), which corresponds to an approximately 7-fold change in the amount of the starting target, because the PCR efficiency was found to be close to 105% [20], which corresponds to an amplification rate of 2.05 per cycle. Thus, with this assay, slight n-fold expression changes of below 7 cannot be distinguished from differences caused by degradation.…”

“…cDNA from post-mortem human brain, cardiac muscle and skeletal muscle tissue was diluted with HPLC grade water (VWR International, Fontenay sous Bois, France) to a concentration equivalent to 5 ng/μL of total RNA, whereas cDNA generated from accurately degraded commercial human brain, heart and skeletal muscle total RNA had a final concentration equivalent to 10 ng/μL of RNA. For all samples, the transcript amount of beta-actin (ACTB, Hs00357333_g1), beta-2-microglobulin (B2M, Hs00187842_m1) and 18S rRNA (Hs99999901_s1, all from Applied Biosystems) was detected using real-time PCR as described previously [20]. For the 18S rRNA assay, an additional dilution step of 1 to 10 was necessary prior to qPCR.…”

“…In addition to the data generated in the present study, data published previously were reanalysed: Non-normalised C q values obtained from RT-qPCR of three gene transcripts (B2M, ACTB and 18S rRNA) in skeletal muscle, cardiac muscle and brain tissue cDNA of 37 individuals, as well as the associated calibrated normalised relative quantities (CNRQ values) calculated using qBasePlus software [23] in Koppelkamm et al [20], were reanalysed to determine whether a data normalisation step can eliminate the influence of impaired RNA integrity. The statistical correlation between the RNA integrity number and C q values or CNRQ data, respectively, was determined by Pearson correlation (R).…”

“…Samples were homogenized in denaturing solution with an IKA-T10 homogenizer (IKA, Staufen, Germany) and finally dissolved in 100 μL nuclease-free water. DNase treatment and additional RNA cleanup were performed as described previously [20]. To minimise possible nuclease-mediated RNA degradation during experimental workflow, all materials and working surfaces were cleaned using RNase Away (Molecular Bioproducts Inc., San Diego, CA, USA).…”

“…Additionally, the individual parameters "age at death" and "body mass index" were included in the present study. Finally, we aimed to analyse whether a carefully validated normalisation strategy as described previously [20] is suitable to balance the influence of low RNA integrity on RT-qPCR results.…”