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PCR Methylation specific PCR

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Get tips on using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® to perform RNA sequencing Rat - PC12

Products New England BioLabs NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®

Get tips on using STEAP2 metalloreductase to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) STEAP2

Products Thermo Fisher Scientific STEAP2 metalloreductase

Get tips on using SIRT1 siRNA and shRNA Plasmids (h) to perform siRNA / miRNA gene silencing Human - PC3 (human prostate cancer cell line) SIRT1

Products Santa Cruz Biotechnology SIRT1 siRNA and shRNA Plasmids (h)

Get tips on using STEMdiff™ Trilineage Differentiation Kit to perform Stem cell Differentiation media Differentiation of Human primed induced pluripotent stem cells (UMN PCBC16iPS) into naive pluripotent stem cells

Products Thermo Fisher Scientific STEMdiff™ Trilineage Differentiation Kit

Get tips on using SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs to perform Cell line authentication Human prostatic cancer cell line PC3

Products Fluidigm SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs

Transfection is a powerful technique that enables the study of the function of genes and gene products in cells. Based on the nature of experiments, we may need a stable DNA transfection in cells for persistent gain-of-function or loss-of-function of the target gene. For stable transfection, integration of a DNA vector into the chromosome is crucial which requires selective screening and clonal isolation. By carefully selecting a viral delivery system and related reagents we can ensure safe and highly-efficient delivery of expression constructs for high-level constitutive or inducible expression in any mammalian cell type.

DNA DNA transfection Mammalian cells Immortalized cell lines PC12

Get tips on using MISSION® pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles to perform shRNA gene silencing Human - Islets of langerhans Negative control (scrambled) lentiviral particles

Products Sigma-Aldrich MISSION® pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include: 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi have been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining efficacy of transduction and shRNA on its target site.

RNA shRNA gene silencing Mouse Prostate cancer cell lines (DU145 and PC3) CD24 lentiviral particles

Get tips on using Gentra Puregene Yeast/Bact. Kit to perform DNA isolation / purification Yeast - Candida parapsilosis

Products Qiagen Gentra Puregene Yeast/Bact. Kit

Get tips on using High Pure RNA Isolation Kit to perform RNA isolation / purification Bacteria - Gram negative Vibro parahaemolyticus

Products Roche Lifesciences High Pure RNA Isolation Kit

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