Methods in molecular biology
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Staging is one of the most important factors that determine the prognosis of the patients with esophageal adenocarcinoma. The current staging protocol includes parameters-extent of tumor (T), lymph node status (N), presence of distant metastasis (M), and grade of the adenocarcinoma (G). In addition, different criteria are used in patients with and without neoadjuvant therapy. The silent aspects of the use of new staging protocol are discussed.
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Single-cell transcriptome sequencing, often referred to as single-cell RNA sequencing (scRNA-seq), is used to measure gene expression at the single-cell level and provides a higher resolution of cellular differences than bulk RNA-seq. With more detailed and accurate information, scRNA-seq will greatly promote the understanding of cell functions, disease progression, and treatment response. ⋯ Particularly, we present a protocol to discover and validate cancer stem cells (CSCs) using scRNA-seq. Suggestions have also been made to help researchers rationally design their scRNA-seq experiments and data analysis in their future studies.
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Pathological assessment of tissue is the gold standard for diagnosis and staging of neoplasia and provides key prognostic information for clinical management. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Endoscopic mucosal resection is a technique used for removing early neoplastic glandular lesions of the esophagus at the level of submucosa. Here, we describe the macroscopic assessment and dissection techniques used for the routine handling of endoscopic mucosal resection specimens in the clinical laboratory.
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Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. ⋯ Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.
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Prospective or "de novo" biobanking is becoming increasingly popular. Biobanks are installed to provide large collections of biological materials for future medical research. ⋯ Therefore, it is vital that all samples are collected and processed in a similar manner according to standardized procedures to ensure high-quality samples and reduce variability in the analytical process. We describe the processes of the centralized biobanking facility at the Leiden University Medical Center (LUMC).