Rapid Isolation of Total RNA from Small Samples of Hypocellular, Dense Connective Tissues

Reno, Carol; Marchuk, Linda L.; Sciore, Paul; Frank, Cyril B.; Hart, David A.

doi:10.2144/97226bm16

Cited by 282 publications

(218 citation statements)

References 2 publications

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“…We performed extraction of total RNA using the TRIspin method [14], which is a combination of the Trizol method and RNAeasy Total RNA Kit (Qiagen, MERCK Eurolab, Stockholm, Sweden). We placed the frozen tendons one by one in a liquid-nitrogen-cooled vessel and pulverized using a Retsch Mixer Mill MM 200 (Retsch GmbH, Haan, Germany).…”

Section: Methodsmentioning

confidence: 99%

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Eliasson

Fahlgren

Aspenberg

2008

Clinical Orthopaedics &Amp; Related Research

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Healing of the rat Achilles tendon is sensitive to mechanical loading, and the callus strength is reduced by 3 = 4 after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This implies BMP signaling is crucial during tendon healing and influenced by mechanical loading. We therefore asked if mechanical loading influences the gene expression of the BMP signaling system in intact and healing tendons, and how the BMP signaling system changes during healing. The genes were four BMPs (OP-1/BMP-7, GDF-5/CDMP-1/BMP-14, GDF-6/CDMP-2/BMP-13, and GDF-7/CDMP-3/BMP-12), two receptors (BMPR1b and BMPR2), and the antagonists follistatin and noggin. The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. All genes except noggin were expressed at all time points, but followed different patterns during healing. Loading strongly decreased the expression of follistatin, which could lead to increased signaling. The BMP system appears involved in tendon maintenance and healing, and may respond to mechanical loading.

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Section: Methodsmentioning

confidence: 99%

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Eliasson

Fahlgren

Aspenberg

2008

Clinical Orthopaedics &Amp; Related Research

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“…Total RNA was extracted from samples using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA was quantified using Ribogreen, and reverse transcription was performed using the Omniscript RT Kit (Qiagen, Mississauga, ON, Canada) (28) . The same number of samples for each cDNA was analysed to prevent bias in the results.…”

Section: Real-time Pcrmentioning

confidence: 99%

High-fat, sucrose diet impairs geometrical and mechanical properties of cortical bone in mice

et al. 2009

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Exposure to diets high in fat and sucrose can induce hyperinsulinaemia, affect Ca and Mg metabolism, and alter bone mineralisation and mechanical properties. The present study assessed morphological and mechanical changes in a murine model exposed to a high-fat/sucrose (HFS) diet, as well as corresponding molecular and endocrine markers of bone turnover. Female C57BL/6 mice (aged 9 weeks) consumed either a low-fat, complex carbohydrate diet or an HFS diet for 10 weeks. At the end of the 10 weeks, serum was collected for biochemical analysis. Tibiae from half the mice (n 15) were randomly selected to be micro-computed tomography scanned and tested to failure in cantilever bending, while the remaining half were prepared for real-time PCR analysis. Serum tartrate-resistant acid phosphatase was significantly elevated in HFS mice, while osteocalcin remained unchanged. Both body mass and percentage body fat were greater in mice fed HFS diet. After adjusting for body mass, tibial structural and morphological properties were adversely affected in the HFS cohort. Cortical thickness, cross-sectional area, and load at maximum were all significantly lower in mice fed HFS diet. Receptor activator of nuclear factor kb ligand (RANKL) mRNA was significantly upregulated in HFS mice, but osteoprotegerin/RANKL mRNA ratio remained unchanged between cohorts. Moreover, cyclo-oxygenase-2 mRNA tended to be increased in HFS. Thus, ingestion of an HFS diet had a significant adverse effect on mouse bone morphology and mechanics, and these effects were likely due to elevated osteoclast activity associated with the inflammatory state of obesity, and not necessarily osteoclast recruitment/ proliferation.Cortical bone: High-fat/sucrose diet: Biomechanics: C57BL/6 mice The changing face of disease aetiology over the past century has brought to the forefront the need to examine the environmental or modifiable risk factors that have influenced the epidemic of chronic disease afflicting a majority of the Western world. Nutritional status is a readily modifiable risk factor linked to numerous chronic conditions, including CHD, cancer, obesity, diabetes and osteoporosis (1,2) . Nonetheless, despite the prevalence of research linking poor dietary choices to chronic disease status, the nutritional patterns presently dominating most Western nations continue to be high in saturated fats, Na and refined carbohydrates from sugars, cereals and dairy products, and low in fruits, vegetables and lean protein (3) .Considerable effort has focused on discovering the underlying nutritional factors that predispose an individual to osteopenia or osteoporosis and subsequent fracture (4) . Much research has concentrated on the role of Ca and vitamin D metabolites influencing bone morphology and mechanics; however, macronutrients such as fat, protein and carbohydrate also play a vital role in bone health (5) . Considered independently, consumption of diets composed of large amounts of saturated fat has been documented to cause the formation of insoluble soap complexe...

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“…method published by Reno et al 1997 . A rotor᎐ stator homogeniser was used to disrupt tendon tissue samples in 1 ml of home-made monophasic reagent containing phenol, isoamyl alcohol, guanidinium Ž isothiocyanate and beta-mercaptoethanol Weber et .…”

Section: Rna Extraction and Quantificationmentioning

confidence: 99%

Multiple changes in gene expression in chronic human Achilles tendinopathy

et al. 2001

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TMŽ . Atlas cDNA cell interaction arrays CLONTECH were used to examine degenerate tissue from four patients with Achilles tendon disorders, in order to identify changes in expression of genes important in cell᎐cell and cell᎐matrix Ž . interactions. The greatest difference between normal post-mortem and degenerate tissue samples was in the level of MMP-3 Ž . stromelysin mRNA, which was down-regulated in all the degenerate samples. Quantitative RT-PCR assay of RNA extracted from paired 'normal' and degenerate Achilles tendon tissue samples taken from tendons during surgery mirrored the results of the arrays. Levels of MMP-3 mRNA were lower, whereas levels of type-I and type-III collagen mRNAs were significantly higher, in the degenerate compared to the 'normal' samples. Immunoblotting of proteins extracted from the same tendon samples showed that three of four normal tissue samples taken from individuals without apparent tendon disorder had much higher levels of MMP-3 protein than 'normal' or degenerate samples from patients with tendinosis. We suggest that MMP-3 may play an important role in the regulation of tendon extracellular matrix degradation and tissue remodelling. ᮊ

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Rapid Isolation of Total RNA from Small Samples of Hypocellular, Dense Connective Tissues

Cited by 282 publications

References 2 publications

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

Mechanical Load and BMP Signaling During Tendon Repair: A Role for Follistatin?

High-fat, sucrose diet impairs geometrical and mechanical properties of cortical bone in mice

Multiple changes in gene expression in chronic human Achilles tendinopathy

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