Abstract
Quantitative polymerase chain reaction (qPCR) is the most specific and reliable method for determination of mRNA gene expression. Crucial point for its accurate normalization is the choice of appropriate internal control genes (ICGs). In the present work we determined and compare the expression of eight commonly used ICGs in lymphocytes from 26 patients with amyotrophic lateral sclerosis (ALS) and 30 control subjects. Peripheral blood mononuclear cells (PBMCs) before and after immortalization by EBV transfection (lymphoblast cell lines—LCLs) were used for qPCR analysis. LCLs were studied before and after liquid nitrogen cryopreservation and culturing (groups LCL1 and LCL2, respectively). qPCR data of 8 ICGs expression was analyzed by BestKeeper, NormFinder and geNorm methods. All studied genes (18SRNA, ACTB, B2M, GUSB,GAPDH, HPRT1, MT-ATP6 and RPS17) were expressed in PBMCs, whereas only first four in LCLs. LCLs cryopreservation had no effect on ICGs expression. Comprehensive ranking indicated RPS17 with MT-ATP6 as the best ICGs for qPCR in PBMCs of control and ALS subjects, and RPS17 with 18RNA or MT-ATP6 in LCLs from ALS. In PBMCs 18RNA shouldn’t be used as ICG.
Highlights
Amyotrophic lateral sclerosis (ALS) is a rare, incurable and fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons, causing skeletal muscle weakness, wasting and death within 3–5 years from the first symptom onset [1]
The studies were conducted on peripheral blood mononuclear cells (PBMC) from 26 ALS patients diagnosed at the Department of Neurology, Medical University of Warsaw (13 women and 13 men), and from 30 age- and gender-matched control individuals without neoplastic and neurodegenerative disorders (14 women and 16 men)
All studied genes were expressed in PBMCs of control and ALS cases and used for validation
Summary
Amyotrophic lateral sclerosis (ALS) is a rare, incurable and fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons, causing skeletal muscle weakness, wasting and death within 3–5 years from the first symptom onset [1]. The number of cases newly diagnosed with ALS each year is 1–3 per 100,000. About 90% of all diagnosed cases are sporadic (SALS) mostly of unknown origin, and the remaining 10% of individuals, with at least one other family member affected, have familial (FALS) [2]. Definitive diagnosis is difficult in early stages of ALS and its confirmation requires a period of observation [3]. There is no single, specific biochemical marker of ALS. Genetic testing may be helpful in diagnosis of FALS and its discrimination from SALS. Peripheral blood mononuclear cells (PBMCs) and human immortalized lymphoblast cell lines (LCLs) derived from PBMCs are a good and long-lasting source of nucleic acids for this type of studies
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