Skeletal muscle biopsy
R. C. Richard Davison, Paul M. Smith, James Hopker, Michael J. Price, Florentina Hettinga, Garry Tew, Lindsay Bottoms in Sport and Exercise Physiology Testing Guidelines: Volume I – Sport Testing, 2022
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.
Microarrays: Human Disease Detection and Monitoring
Attila Lorincz in Nucleic Acid Testing for Human Disease, 2016
A preanalytical study by Mutter et al. evaluated the effects of three different tissue storage methods (fresh, frozen and preserved with RNALater*) on the same uterine myometrial tissue specimen by comparing quantitative microarray expression.85 The group used analysis of variance (ANOVA) (http://www.physics.csbsju.edu/stats/anova.html) and found very little expression variation among the three cases. When compared by gene functional class, the frequency of outliers was overall no more than what was expected by random chance. This study suggests that if tissue is maintained in a stable environment, for example, via RNALater treatment or snap freezing, a number of methods can be used as a means of routine tissue collection and temporary storage prior to RNA extraction.
Pathology in the Era of Personalized Medicine
II-Jin Kim in 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].
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.
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).
Perivascular extension of microwave ablation zone: demonstrated using an ex vivo porcine perfusion liver model*
Published in International Journal of Hyperthermia, 2018
Saurabh Singh, Pulathis Nilantha Siriwardana, Edward William Johnston, Jennifer Watkins, Steven Bandula, Rowland Illing, Brian Ritchie Davidson
In order to assess histological evidence of tissue ablation along the length the vessels, one half of the bisected specimen was fixed in formalin and paraffin embedded with the mid sagittal plane facing up. Five consecutive 4 µm sections from the side of the mid sagittal plane were obtained and mounted on charged glass slides. These were stained with Hematoxylin and Eosin (H&E). In order to obtain cryosections, which corresponded with the paraffin sections, a 5 mm slice was obtained from the remaining half of the bisected ablation zone. These were mounted fresh with the mid sagittal plain facing up, snap frozen in liquid nitrogen and stored at –80 °C. The method of bisecting the ablation zone and obtaining a section snap freezing is shown in diagrammatic form in Figure 3. Cell viability in areas of coagulation necrosis following thermal ablation was evaluated using histochemical staining for nicotinamide adenine dinucleotide (NADH) as previously described [18]. NADH is a ubiquitous coenzyme present in both cytoplasm and mitochondria of viable cells. A validated protocol was used for NADH staining [19]. A blue formazan deposit in the nuclei as well as the cytoplasm indicates cellular enzyme activity; hence cell viability at the time of tissue preservation.
Related Knowledge Centers
- Pasteurization
- Protein
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- Liquid Nitrogen
- Cell
- Flash Freezing
- Blast Chilling