Motivation:e scratch assay is a standard experimental protocol used to characterize cell migration. It can be used to identify genes that regulate migration and evaluate the e cacy of potential drugs that inhibit cancer invasion. In these experiments, a scratch is made on a cell monolayer and recolonisation of the scratched region is imaged to quantify cell migration rates. A drawback of this methodology is the lack of its reproducibility resulting in irregular cell-free areas with crooked leading edges. Existing quanti cation methods deal poorly with such resulting irregularities present in the data. Results: We introduce a new quanti cation method that can analyse low quality experimental data. By considering in-silico and in-vitro data, we show that the method provides a more accurate statistical classi cation of the migration rates than two established quanti cation methods. e application of this method will enable the quanti cation of migration rates of scratch assay data previously unsuitable for analysis. Availability and Implementation: e source code and the implementation of the algorithm as a GUI along with an example dataset and user instructions, are available in https://bitbucket.org/ anavictoria-ponce/local migration quantification scratch assays/src/ master/. e datasets are available in https://ganymed.math.uni-heidelberg.de/ ∼victoria/publications.shtml.
BackgroundMolecular and cellular pathophysiological events occurring in the majority of rare kidney diseases remain to be elucidated. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive disorder caused by mutations in either CLDN16 or CLDN19 genes. This disease is characterized by massive urinary wasting of magnesium and calcium, osmosis deregulation and polyuria. Patients with p.G20D homozygous mutation in CLDN19 gene exhibit different progression to kidney failure suggesting that beyond the pathogenic mutation itself, other molecular events are favoring disease progression. Due to the fact that biopsy is not clinically indicated in these patients, urinary exosome-like vesicles (uEVs) can be envisioned as a valuable non-invasive source of information of events occurring in the kidney. Exosome research has increased notably to identify novel disease biomarkers but there is no consensus standardized protocols for uEVs isolation in patients with polyuria. For this reason, this work was aimed to evaluate and refine different uEVs isolation methods based on differential centrifugation, the gold standard method.ResultsCharacterization by NTA, cryo-TEM and immunoblotting techniques identified the most appropriate protocol to obtain the highest yield and purest uEVs enriched fraction possible from urine control samples and FHHNC patients. Moreover, we tested five different RNA extraction methods and evaluated the miRNA expression pattern by qRT-PCR.ConclusionsIn summary, we have standardized the conditions to proceed with the identification of differentially expressed miRNAs in uEVs of FHHNC patients, or other renal diseases characterized by polyuria.
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