Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity

Vrtačnik, Peter; Kos, Špela; Bustin, Stephen A.; Marc, Janja; Ostanek, Barbara

doi:10.1016/j.ab.2014.06.017

Cited by 16 publications

(14 citation statements)

References 28 publications

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“…For cell dissociation, we have used papain only for hippocampal cells rather than trypsin, while cortical cells were dissociated by trituration without any prior enzymatic digestion. It has been observed that trypsin digestion of tissue leads to RNA degradation [20]. The cortical cells were allowed to recover from mechanical dissociation through a short incubation at 37 • C in DMEM.…”

Section: Discussionmentioning

confidence: 99%

Culturing primary neurons from rat hippocampus and cortex

et al. 2019

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Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods.

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Section: Discussionmentioning

confidence: 99%

Culturing primary neurons from rat hippocampus and cortex

et al. 2019

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“…For cell dissociation we have used papain only for hippocampal cells rather than trypsin, while cortical cells were dissociated by trituration without any prior enzymatic digestion. It has been observed that trypsin digestion of tissue leads to RNA degradation (16). The cortical cells recover from mechanical dissociation through a short incubation at 37 °C in DMEM with serum.…”

Section: Discussionmentioning

confidence: 99%

Culturing primary neurons from rat hippocampus and cortex

Sahu

Nikkilä

Lågas

et al. 2018

Preprint

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7Primary neurons from rodent brain hippocampus and cortex has served as great tools in biomedical 8 research over the years. However, protocols for the preparation of primary neurons vary, which often 9 leads to conflicting results. This report provides a robust and reliable protocol for the production of 10 primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-11 neuronal cells. The neurons were grown in serum free media and maintained for several weeks 12 without any additional feeder cells. The neuronal cultures maintained according to this protocol 13 differentiate and by three weeks develop extensive axonal and dendritic branching. The cultures 14 produced by this method show excellent reproducibility and can be used for histological, molecular 15 and biochemical methods. 16 17

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“…For studies involving RNA investigation, the introduction of trypsinization as part of the experimental process results in a complete degradation of RNA regardless of cell line type, differentiation stage, or passage number. 59 Thus, findings from RNA studies may not rely on high/low dichotomization but rather on trypsin as present, and therefore acting as a modifier or catalyst of other processes that drive further proliferation and invasive process in pancreatic cancer.…”

Section: Why Has Trypsin Yet To Be Investigated As a Major Player Befmentioning

confidence: 99%

Is There a Trojan Horse to Aggressive Pancreatic Cancer Biology? A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis

Søreide

Roalsø

Aunan

2020

Journal of Pancreatic Cancer

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Purpose: Pancreatic cancer is one of the most lethal of solid tumors and is associated with aggressive cancer biology. The purpose is to review the role of trypsin and effect on molecular and cellular processes potentially explaining the aggressive biology in pancreatic cancer. Methods: A narrative literature review of studies investigating trypsin and its effect on protease systems in cancer, with special reference to pancreatic cancer biology. Results: Proteases, such as trypsin, provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Trypsin is a digestive enzyme produced by the exocrine pancreas that is also related to mechanisms of proliferation, invasion and metastasis. Several of these mechanisms may be co-regulated or influenced by activation of proteinaseactivated receptor 2 (PAR-2). The current role in pancreatic cancer is not clear but emerging data suggest several potential mechanisms. Trypsin may act as a Trojan horse in the pancreatic gland, facilitating several molecular pathways from the onset, which leads to rapid progression of the disease. Pancreatic cancer cell lines containing PAR-2 proliferate upon exposure to trypsin, whereas cancer cell lines not containing PAR-2 fail to proliferate upon trypsin expression. Several mechanisms of action include a proinflammatory environment, signals inducing proliferation and migration, and direct and indirect evidence for mechanisms promoting invasion and metastasis. Novel techniques (such as organoid models) and increased understanding of mechanisms (such as the microbiome) may yield improved understanding into the role of trypsin in pancreatic carcinogenesis. Conclusion: Trypsin is naturally present in the pancreatic gland and may experience pathological activation intracellularly and in the neoplastic environment, which speeds up molecular mechanisms of proliferation, invasion, and metastasis. Further investigation of these processes will provide important insights into how pancreatic cancer evolves, and suggest new ways for treatment.

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Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity

Cited by 16 publications

References 28 publications

Culturing primary neurons from rat hippocampus and cortex

Culturing primary neurons from rat hippocampus and cortex

Culturing primary neurons from rat hippocampus and cortex

Is There a Trojan Horse to Aggressive Pancreatic Cancer Biology? A Review of the Trypsin-PAR2 Axis to Proliferation, Early Invasion, and Metastasis

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