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Skeletal muscle biopsy
Published in R. C. Richard Davison, Paul M. Smith, James Hopker, Michael J. Price, Florentina Hettinga, Garry Tew, Lindsay Bottoms, Sport and Exercise Physiology Testing Guidelines: Volume I – Sport Testing, 2022
Richard A. Ferguson, Natalie F. Shur
Muscle tissue can be prepared in several ways depending on the subsequent analysis. For measurement of muscle metabolites, gene expression, mRNA/protein content and enzyme activity, the tissue is quickly snap-frozen in liquid nitrogen and subsequently stored at −80°C until analysis. In the case of analysis of high-energy phosphates, the time taken between the muscle sampling and snap freezing should be rapid (and can be as quick as 3 sec; Hultman and Sjöholm, 1983). One may perform subsequent assays on homogenised samples. One may also conduct fibre-type specific analysis on single fibres micro-dissected from freeze-dried samples. Despite being very time consuming, one section of the dissected fibre is analysed for fibre type using acid-labile myofibrillar ATPase histochemistry (Essen et al., 1975), SDS-PAGE for myosin heavy-chain isoform determination (Sant’Aana Pereirra et al., 1995) or, more recently, a dot blotting method based on Western blotting techniques (Christiansen et al., 2019). The remaining fibre fragment, or pooled samples of the same fibre type, are then analysed for the metabolites, gene transcripts or proteins of interest.
Acquisition and Preservation of Tissue for Microarray Analysis
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Sample handling is an important step that can lead to variability observed in microarray results. Ischemia time can profoundly affect protein, DNA, and RNA extraction because of enzymatic degradation. As noted above, sample collection is ideally performed by snap-freezing tissue in liquid nitrogen immediately after being devascularized and removed from a patient. Sample handling of formalin-fixed, paraffin-embedded tissue is variable, and this makes application of the array data potentially unreliable.
Pathology in the Era of Personalized Medicine
Published in II-Jin Kim, Cancer Genetics and Genomics for Personalized Medicine, 2017
The accuracy and reproducibility of molecular diagnosis depend on the quantity and quality of the cancer tissue specimens. Although snap freezing of tumor tissues in liquid nitrogen is the optimal method for preserving nucleic acids, in many pathologic laboratories, tumor tissues usually are formalin-fixed and paraffin-embedded (FFPE) to preserve histological features. The nucleic acids and proteins in tissue specimens are affected by the type of fixative used, the duration of fixation, decalcification, and the storage conditions (i.e., time, temperature, and humidity) [11]. The recommended method is to quickly transfer the specimen into 10% neutral buffered formalin, usually within 1 hour, and to fix the specimen for 8–72 hours [6, 12]. Both prolonged preanalytic cold ischemia time and overfixation or underfixation can cause false-negative or invalid results. In addition, the storage conditions and duration of storage for the FFPE blocks can affect test results. Even if the tumor tissue is frozen, the nucleic acids degenerate and become fragmented with after several years of storage [11].
GSPE pre-treatment protects against long-term cafeteria diet-induced mitochondrial and inflammatory affectations in the hippocampus of rats
Published in Nutritional Neuroscience, 2022
Oriol Busquets, Marina Carrasco, Triana Espinosa-Jiménez, Miren Ettcheto, Ester Verdaguer, Carme Auladell, Mònica Bullò, Antoni Camins, Montserrat Pinent, Esther Rodríguez-Gallego, Jaume Folch
The quantification of transcriptional activity was determined through RT–PCR. All details for RNA extraction, retrotranscription into cDNA and plate preparation methods were reported in a previous publication from our research group [19]. Briefly, starting from the previously mentioned protocol in section 2.2, RNA fraction was obtained by using the TriSure RNA extraction protocol (Bioline; BIO-38032). Samples were stored by snap freezing them in dry ice and keeping them in −80C conditions until use. Retrotranscription was performed by using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher; 4368814). cDNA samples were kept in −20C conditions. Plate preparation was done following the guidelines indicated by the SYBR Green provider (ThermoFisher). Gapdh was used as housekeeping gene. ST (n =4), CAF (n =4) and CAF+500 (n =4).
Preformulation studies of thymopentin: analytical method development, physicochemical properties, kinetic degradation investigations and formulation perspective
Published in Drug Development and Industrial Pharmacy, 2021
Mengyang Liu, Darren Svirskis, Thomas Proft, Jacelyn Mei San Loh, Jingyuan Wen
TP5-ME displays a spherical structure with the unilamellar vehicle property as shown in the Cryo-TEM graph (Figure 9(a)). The diameter of TP5-ME exhibits around 150 nm, which is consistent with the result from droplet size analysis. The nanostructure and lamellarity of TP5-ME were also observed using Cryo-SEM as shown in Figure 9(b). Wherein, some aggregated droplets were observed, which may explain the second highest peak of 205 nm in previous size distribution study. The micrograph captured confirms the spherical-shaped ME and typical concentric monolayer. The rough surfaces of ME were contributed to the sample preparation technique for Cryo-SEM which is based on snap freezing in liquid nitrogen. It also can be verified that the size distribution is matched to Nanosight® data.
Gene expression-based biodosimetry for radiological incidents: assessment of dose and time after radiation exposure
Published in International Journal of Radiation Biology, 2019
Ellina Macaeva, Mohamed Mysara, Winnok H. De Vos, Sarah Baatout, Roel Quintens
In addition, finding the right methodological approach to monitor gene expression as early as possible following exposure is also important because the signal is lost within days (Hall J et al. 2017). Possible solutions to this include immediate snap freezing of blood in liquid nitrogen or dry ice, which might be challenging in field conditions, or addition of special whole blood preservation buffers (Schwochow et al. 2012), which would also solve the problem of effective preservation of easily-degradable RNA. Another challenge of using whole blood for gene expression studies is the heterogeneity of blood cells. About 99% of blood cells are red blood cells, including immature reticulocytes, which contain high levels of globin mRNA accounting for ∼70% of all mRNA in whole blood. This can compromise the detection of other specific mRNAs from white blood cells (Field et al. 2007). Although qPCR is less affected by globin mRNA contamination, this parameter is highly important for such techniques as microarrays (Liu et al. 2006) and next-generation sequencing (Schwochow et al. 2012).