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

The International Frontal Sinus Anatomy Classification (IFAC) and Classification of the Extent of Endoscopic Frontal Sinus Surgery (EFSS)

The frontal recess and frontal sinus anatomy can vary from simple to complex. The variations in the anatomy of the frontal recess and frontal sinus are considerable but almost all variations can be classified if the various cell patterns are analyzed. This consensus document was developed to improve the ability of the surgeon to understand these possible variations, plan the surgery, and communicate these complexities when teaching or reporting outcomes. Once the surgeon understands the anatomical pattern of the frontal sinus and recess cells, the extent of surgery can be planned. This document presents a classification of the extent of surgery based on the anatomical classification. (C) 2016 ARS-AAOA, LLC