Source: Journal of Neuroscience Methods, Paperback 209, Issue 1
Jorge H. Limon-Pacheco, Jana Mejía-Toiber, Adriana Gonzalez-Gallardo, Magda Giordano
Plasmid retention after long-term transplant was a the major technical limitations for transplantation studies. This search will find the use of a modified protocol of Hirt and SYBR Green-based quantitative PCR period (qPCR) and to determine a vector containing a peculiar transgene in transfected cells after transplantation brain.We compared all the different methods for sample preparation and utilization of extra-chromosomal DNA suitable for qPCR.Die modified protocol of Hirt, the record was reliable, optimal plasmid recovery from transplanted tissue with minimal destruction of plasmid DNA, compared with a commercial kit or TRIzol ® protocols. The PCR protocol for the recording and all transgene detection built an image of two very specific primer sets to capture the sequence of human glutamic acid decarboxylase 1 (hGAD67) transgene by SYBR Green-based qPCR, and the existence of the vector pREP10 hGAD67 confirmed by endpoint PCR.We used a standard curve of subsequent dilution of pure plasmid pREP10 hGAD67 As mentioned in qPCR experiments, obtained the number of plasmid copies from cultured cells and tissue samples according to Hirt extraction find formed ist.Historische, plasmid stability was evaluated in transplanted tissues after different time intervals, and the plasmid loss in the body part of interest, it was found that, for example babelike.In this study, we describe a simple, highly specific, inexpensive and reliable system for plasmid recovery and quantification of the transgene of interest in long-term brain transplant studies, this method can be very extensive to other models of transplantation.
► We do all three methods compared for plasmid seizure of transplanted cells and brain. ► a modified protocol of Hirt was optimal for plasmid recovery of transplanted tissue. ► plasmids by modified rule of shepherd is suitable for all SYBR Green PCR-based.► plasmid is gradually lost in a time dependent manner. ► We are an inexpensive and reliable method for quantification of plasmid in transplant call.