The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown,
respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]
Standard operating procedure for the determination of tissue fungal burden utilizing quantitative real time polymerase chain reaction (QPCR). This standard operating procedure will provide information on how to assess fungal tissue burden
of infected animals by use of a single copy (FKS) or multicopy gene (18s RNA) to assess the number of fungal cell nuclei present. - [Read Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction]
This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon (Transgenic Res. 12, 521-522 (2003)).For those familiar with PCR genotyping, this method differs from the typical protocol by utilizing a unique enzyme (Klentaq), 30mer primers, and a 68° annealing temperature. - [Read Universal Mouse Genotyping Protocol]