Site Directed Mutagenesis (SDM) Human Point mutation THP-1

Get tips on using ON-TARGETplus Human ABL2 (27) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 ARG/ABL2

Get tips on using ON-TARGETplus Human RAB11FIP1 (80223) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - H1299 Rab Coupling Protein (RCP)

Get tips on using ON-TARGETplus Human RAB11FIP1 (80223) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - A431 Rab Coupling Protein (RCP)

Get tips on using ON-TARGETplus Human ITGB3 (3690) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 β3 integrin/ITGB3

Get tips on using ON-TARGETplus Human ITGB1 (3688) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - MDA-MB-231 β1 integrin/ITGB1

Get tips on using ON-TARGETplus Human LIN28A (79727) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Human - hES cell line H1 (WA01) LIN28

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.