Formalin-fixed paraffin-embedded (FFPE) tissue specimens have been a staple of research, providing precious resources for molecular and genomic studies. However, the biggest challenge is the extraction of high-quality DNA from FFPE tissues, given that the integrity of DNA is critically affected by formalin fixation. Formaldehyde induces crosslinks in DNA that renders single or double-stranded DNA breaks. Such breaks cause extensive fragmentation that directly influences the quality of DNA purified and the number of templates available for PCR amplification. Thus, protocol for DNA purification from FFPE tissues must effectively extract highly fragmented DNA and reverse cross-linking caused by formalin fixation. DNA extraction methods available in the literature were selected and modified at different stages to optimize a protocol that extracts DNA of sufficient quality and fragment size to be detectable by PCR. Archived FFPE tissues belonged to patients with triple negative breast cancer (TNBC) and benign breast disease were used for the protocol optimization. The best optimized protocol was then used to amplify Exon 4 region of Proviral integration site for Moloney murine leukemia virus1 (Pim1) kinase gene to analyze any probable somatic mutations both in TNBCs and benign breast diseases. Of the 12 different protocols developed, best quality DNA in terms of fragment size and purity was obtained when Tween20 lysis buffer was used for both deparaffinization and overnight digestion along with high salt precipitation. Optimized protocol was then validated by extracting DNAs from 10 TNBCs and 5 benign breast disease specimens with consistent purity and fragment size. PCR amplification and subsequent Sanger's sequencing revealed the presence of mutations in the Exon 4 region of Pim1 kinase. Deparaffinization and overnight digestion in Tween20 lysis buffer along with high salt precipitation yielded the best quality PCR amplifiable DNA for mutational analysis.
Purpose: A chronic wound model provides an opportunity to understand fundamental mechanisms that could possibly provide leads for novel diagnostic molecules. An in vitro chronic wound model was created to evaluate the healing potential of stem cell (SC) secretome obtained from human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and cord blood platelet lysate (CBPL).
Methods: hUCMSCs were obtained from explant culture of umbilical cord. SC secretome was collected from cells at passage 2 after appropriate SC characterisation. Platelets were isolated from cord blood and exposed to a repeated freeze–thaw cycle to obtain CBPL. An in vitro chronic wound model was created using dimethyl sulfoxide (DMSO) and hypoxia in HEK293 cells through scratch in a cell monolayer. Wounds were exposed to SC secretome and CBPL and their wound closure efficacy was calculated as relative wound density (RWD%) at defined time points.
Results: MSCs with typical spindle-shaped morphology were isolated from explant culture that sustained their stemness and morphology until up to passage 10. DMSO efficiently impeded the movement of cells into the wound area to generate a chronic wound model which was efficiently removed upon addition of SC secretome and CBPL.
Conclusion: A transient in vitro chronic wound model was created successfully to determine the healing efficiency of SC secretome and CBPL.
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