embryo-transfer-surgery-via-laparotomy-in-gilts

Embryo Transfer Surgery via Laparotomy in Gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...

Universidade de São Paulo

The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p < 0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5' element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.

Easy In Vitro Synthesis of Optimised Functioning Reporter mRNA from Common eGFP Plasmid.

Histopathology is a well-established standard diagnosis employed for the majority of malignancies, including breast cancer. Nevertheless, despite training and standardization, it is considered operator-dependent and errors are still a concern. Fractal dimension analysis is a computational image processing technique that allows assessing the degree of complexity in patterns. We aimed here at providing a robust and easily attainable method for introducing computer-assisted techniques to histopathology laboratories. Slides from two databases were used: A) Breast Cancer Histopathological; and B) Grand Challenge on Breast Cancer Histology. Set A contained 2480 images from 24 patients with benign alterations, and 5429 images from 58 patients with breast cancer. Set B comprised 100 images of each type: normal tissue, benign alterations, in situ carcinoma, and invasive carcinoma. All images were analyzed with the FracLac algorithm in the ImageJ computational environment to yield the box count fractal dimension (Db) results. Images on set A on 40x magnification were statistically different (p = 0.0003), whereas images on 400x did not present differences in their means. On set B, the mean Db values presented promissing statistical differences when comparing. Normal and/or benign images to in situ and/or invasive carcinoma (all p < 0.0001). Interestingly, there was no difference when comparing normal tissue to benign alterations. These data corroborate with previous work in which fractal analysis allowed differentiating malignancies. Computer-aided diagnosis algorithms may beneficiate from using Db data; specific Db cut-off values may yield ~ 99% specificity in diagnosing breast cancer. Furthermore, the fact that it allows assessing tissue complexity, this tool may be used to understand the progression of the histological alterations in cancer.

Article	Year
Easy In Vitro Synthesis of Optimised Functioning Reporter mRNA from Common eGFP Plasmid. Molecular biotechnology\| PubMed ID: 30120676	2018
Fractal dimension analysis as an easy computational approach to improve breast cancer histopathological diagnosis. Applied microscopy\| PubMed ID: 33929635	2021
Brazilian Guidelines for Cardiac Implantable Electronic Devices - 2023. Arquivos brasileiros de cardiologia\| PubMed ID: 36700596	2023

Gustavo Torres

Biography

JoVE Journal Publications

Other Publications