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Background: RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Results: Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most 'stable' genes and that gene 'stability' is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. Conclusion: To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated "housekeeping" genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution. [ABSTRACT FROM AUTHOR]

In the hair follicle the cuticle develops as a thin layer of cells between the hair shaft cortex and the inner root sheath. Once the cuticle cells begin to differentiate they accumulate cysteine-rich granules in their cytoplasm but the identity of their constituent proteins has remained largely an enigma. In this report we show differential expression of a family of genes encoding cysteine-rich, glycine-rich keratins in the cuticle. Two clones of the sheep KAP5 gene family were isolated: the KAP5.4 cDNA encodes a protein of 190 amino acids (Mr = 16,936) containing 32 mol% cysteine, 26 mol% glycine and the partial KAP5.5 cDNA encodes a protein of at least 197 amino acids (Mr ≥ 17,474) containing 29 mol% cysteine, 28 mol% glycine. The predicted amino acid sequences of the KAP5 family show extensive sequence conservation and all the proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Each KAP5 gene produces an ∼ 1.5- kb mRNA species but the KAP5.4 and KAP5.5 mRNA levels appear to be severalfold greater than the KAP5.1 mRNA. Comparative tissue in situ hybridizations reveal a positive correlation between the onset of expression and follicle depth. For a given KAP5 gene two widely different cuticle expression patterns were noted amongst the follicle populations, and on the basis of follicle bulb cell kinetics they are consistent with expression in either sheep primary or secondary follicle types. [ABSTRACT FROM AUTHOR]

In epithelial differentiation keratin intermediate filament genes are expressed in multifarious tissue-specific and stage-specific patterns. Pairs of type I and type II intermediate filament genes, belonging to multigene families, are coordinately regulated, and 4 - 5 genes of each type are expressed in the hair follicle. Accumulating chromosomal mapping data points to a major locus for each intermediate filament multigene family on separate chromosomes. In this report we describe the isolation of a sheep hair keratin cosmid by chromosome walking that overlaps two previously described cosmids and establishes a continuous 100-kb segment of cloned DNA containing three hair and three hair-like type II intermediate filament keratin genes. A new hair keratin type II intermediate filament gene, KRT2.11, is located in the middle of the cluster, and partial sequence data reveal a striking conservation of its predicted N-terminal region with other sheep hair keratin type II intermediate filament proteins. Expression analyses demonstrate the presence of a 2.4-kb KRT2.11 transcript in wool follicle RNA and show that expression occurs in the follicle cortical keratinocytes above the dermal papilla. The three hair genes are clustered within about 40 kb and flanked by hair-like genes that are not expressed in the hair follicle, thereby demarcating a hair keratin gene domain. [ABSTRACT FROM AUTHOR]

Several families of proteins are expressed in the growth of hair and an estimated 50-100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin. A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para- and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hair-loss mice. Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para- to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought. [ABSTRACT FROM AUTHOR]