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Griffin:siRNA transfection: Difference between revisions
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===Background Info=== | ===Background Info=== | ||
*What are the experimental results? | *What are the experimental results? | ||
*Describe how gene knockdown is measured? | *Describe how gene knockdown is measured? | ||
*How was the RNA reconstituted? | *How was the RNA reconstituted? | ||
'''NOTE:''' siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. | '''NOTE:''' siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution. Having the correct molarity of the solution is critical. | ||
*Molarity of siRNA vialed: 10 uM ( uM/L ) | |||
*Volume after reconstitution: 330 uL | |||
*Mass of 1 mole of siRNA: 13800 g/mol ( 21nt X 660 g/base pair) | |||
*Total mols per vial: 10 um/L X 330 uL = 3.3 nm | |||
*Total grams per vial: 3.3 nm X 13800 g/mol = 45.5 ug | |||
*Solution concentration: 45.5 ug/ 330 uL = 0.138 ug/uL | |||
*Did this same vial or other lot of siRNA work in the past? | *Did this same vial or other lot of siRNA work in the past? | ||
'''NOTE:''' If the siRNA has worked in the past, and now does not work, this may suggest RNase contamination. | '''NOTE:''' If the siRNA same cat# has worked in the past, and now does not work, this may suggest RNase contamination. There are ways to determine this by running 1 pmol (17 ng) siRNA in a native 2% agarose gel, however replacing the vial is a straightforward solution. | ||
===Transfection Efficiency=== | ===Transfection Efficiency === | ||
*Describe the cell type for this experiment? | *Describe the cell type for this experiment? | ||
* | *What transfection reagent is used for the siRNA tranfection? | ||
NOTE: Cationic lipid based transfection reagents (ie Lipofectimine, L2000, Transit TKO, Oligofect, Dharmafect, sc-29528) are each one a unique formula, that certain cell types will respond better to certain cationic lipid (positive charge lipophilic) reagents. For this reason, measuring transfection efficiency is necessary. | |||
*How was transfection efficiency measured? | *How was transfection efficiency measured? | ||
'''NOTE:''' The researcher may have an existing transfection reagent that works on their cells in other experiments (ie cDNA studies), and this is a good indicator to try the same reagent and measure transfection efficiency. | |||
*What time point was transfection uptake of FITC-siRNA measured? | *What time point was transfection uptake of FITC-siRNA measured? | ||
NOTE: Measuring transfection efficiency with sc-36869 will validate that liposome-dependent siRNA entry into the cells is taking place efficiently. It is important to measure transfection efficiency 5-7 hours post transfection since this is when the optimum time point where most transfection takes place. Common methods are IF or Flow cytomtetry. | |||
===Cell Confluency=== | ===Cell Confluency=== | ||
* | *Adherent cell (grows on the surface of the plate): What is the cell confluency at time of transfection? | ||
*Suspension cell (ie leukocytes/lymphocytes, grow in the media) : How many cell count # used to seed the well? | |||
'''NOTE:''' A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. | '''NOTE:''' A hemocytometer (cell counter) is common for counting cells for seeding into multiwell plates (6, 12, 24 well); originally designed for performing blood cell counts. Cell density is an important parameter for knockdown. Optimum cell density will vary and typically falls between 30-80%. | ||
'''NOTE:''' | '''NOTE:''' Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%), will reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%. | ||
===siRNA Concentration=== | ===siRNA Concentration=== | ||
What nanomolar | *What nanomolar concentration(s) of siRNA are tested? | ||
'''NOTE:''' Setting up a 6 or 12 well experiment and trying a range of cell confluencies (30, 50, 70%) & a range of siRNA concentrations (30, 60, 90 nM) will reveal an optimal convergence of cell density and concentration of siRNA where knockdown is optimal with minimal cytotoxicity (cell death). | |||
*What time points is RNAi measured? | |||
'''NOTE:''' Measuring knockdown for a few time points in the 24-72 hour window may indicate the | '''NOTE:''' 48 hours post transfection is a relevant singular point. Measuring knockdown for a few time points in the 24-72 hour window may indicate the frame when RNAi is most optimal. Titrating the siRNA concentration (30-90 nM) for the cells will indicate the best amount to see an effect. | ||
===Measuring Knockdown=== | ===Measuring Knockdown=== | ||
How is RNAi measured? Western blot - IF - qPCR - other | *How is RNAi measured? Western blot - IF - qPCR - other | ||
'''NOTE:''' For WB, titrating the antibody may reveal subtle changes in knockdown. | '''NOTE:''' For WB, titrating the antibody may reveal subtle changes in knockdown. For IF, running secondary controls may indicate nonspecific fluorescence mistaken for signal. | ||
For IF, running secondary controls may indicate nonspecific fluorescence. | |||
Quantitative RT-PCR, which primers were used and what type of system? | *Quantitative RT-PCR, which primers were used and what type of system? | ||
'''NOTE:''' With appropriate internal controls (GAPDH, DNA contamination control), qPCR can be very reliable. | '''NOTE:''' With appropriate internal controls (GAPDH, DNA contamination control), qPCR can be very reliable in determining translation initiation arrest. | ||
==References== | ==References== | ||
