Identification of valid reference genes during the differentiation of human myoblasts

<p>Abstract</p> <p>Background</p> <p>Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.</p> <p>Results</p> <p>Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.</p> <p>Conclusion</p> <p>RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.</p

Multimodal control of neck muscles for vestibular mediated head oscillation damping during walking: a pilot study

Abstract Purpose It is still in question whether head oscillation damping during walking forms a part of the vestibular function. The anatomical pathway from the vestibular system to the neck muscles via the medial vestibulospinal tract (MVST) is well known but there is a lack of knowledge of the exact influence and modulation of each other in daily life activities. Methods (I) We fixed a head–neck unit of a human cadaver specimen in a steal frame to determine the required pitch-torque for a horizontal head position. The mean value of the acquired pitch-torque was 0.54 Nm. (II) On a motorized treadmill we acquired kinematic data of the head, the sternum and both feet by wireless 3D IMUs for seven asymptomatic volunteers. Subsequently three randomized task conditions were performed. Condition 1 was walking without any irritation. Condition 2 imitated a sacculus irritation using a standardized cVEMP signal. The third condition used an electric neck muscle-irritation (TENS). The data were analyzed by the simulation environment software OpenSim 4.0. Results 8 neck muscle pairs were identified. By performing three different conditions we observed some highly significant deviations of the neck muscle peak torques. Analysing Euler angles, we found during walking a LARP and RALP head pendulum, which also was strongly perturbated. Conclusion Particularly the pitch-down head oscillation damping is the most challenging one for neck muscles, especially under biomechanical concerns. Mainly via MVST motor activity of neck muscles  might be modulated by vestibular motor signals. Two simultaneous proprioceptor effects might optimize head oscillation damping. One might be a proprioceptive feedback loop to the vestibular nucleus. Another might trigger the cervicocollic reflex (CCR)

Mutation analysis of "Endoglin" and "Activin receptor-like kinase" genes in German patients with hereditary hemorrhagic telangiectasia and the value of rapid genotyping using an allele-specific PCR-technique

<p>Abstract</p> <p>Background</p> <p>Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant disorder which is clinically characterised by recurrent epistaxis, mucocutaneous telangiectasia and visceral arteriovenous malformations. Genetic linkage studies identified two genes primarily related to HHT: endoglin (<it>ENG</it>) on chromosome 9q33-34 and activin receptor-like kinase1 (<it>ACVRL1</it>) on chromosome 12q13. We have screened a total of 41 unselected German patients with the suspected diagnosis of HHT. Mutation analysis for the <it>ENG </it>and <it>ACVRL1 </it>genes in all patients was performed by PCR amplification. Sequences were then compared to the HHT database <url>http://www.hhtmutation.org</url> sequences of the <it>ENG </it>mRNA (accession no. BC014271.2) and the <it>ACVRL1 </it>mRNA (accession no. NM000020.1).</p> <p>Results</p> <p>We identified 15 different mutations in 18 cases by direct sequencing. Among these mutations, one novel <it>ENG </it>mutation could be detected which has not yet been described in the literature before. The genotype-phenotype correlation was consistent with a higher frequency of pulmonary arteriovenous malformations in patients with <it>ENG </it>mutations than in patients with <it>ACVRL1 </it>mutations in our collective.</p> <p>Conclusion</p> <p>For rapid genotyping of mutations and SNPs (single nucleotide polymorphisms) in <it>ENG </it>and <it>ACVRL1</it>, allele-specific PCR methods with sequence-specific primers (PCR-SSP) were established and their value analysed.</p