Griffin:siRNA transfection: Difference between revisions

From OpenWetWare
Jump to navigationJump to search

Griffin:siRNA transfection (view source)

Revision as of 11:37, 21 July 2010

1,331 bytes added ,  21 July 2010
→‎Troubleshooting
No edit summary
Line 203: Line 203:
==Troubleshooting==
==Troubleshooting==


*Determine your product Catalog # & Lot #
TECHNICAL SERVICE GUIDE: siRNA
*How do you measure knockdown? Western blot, IF or Quantitative RT-PCR?
*What is the issue with the siRNA? 
*What cell type? 
*How was transfection efficiency determined and with what brand transfection reagent? 
Transfection reagents are unqiue and certain cells do respond better to certain reagents. Measuring transfection efficiency with sc-36869 will validate that siRNA is entering the cells efficiently. It is important to always measure transfection efficiency 5-7 hours post transfection since this is when the optimum transfection occurs.


* What cell confluency (adherent) or cell number (suspension) were tested?  
Catalog #          Lot #


Trying a range of cell confluencies from 20-80% may reveal an optimal cell density where knockdown is optimal with minimal cell death.
===Background Info===


*What is the nanomolar siRNA concentration(s) tested?  
*What are the experimental results? 
*Describe how gene knockdown is measured?
*How was the RNA reconstituted?


Measuring 24-72 hour window may indicate the time when kd is most optimal. Titrating the siRNA concentration for the cells may indicate the best amount to see an effect.
NOTE: siRNA ships lyophilized along with RNase free water with instructions to reconstitute with 330 ul of H2O to make 10 uM solution.
 
*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.
 
===Transfection Efficiency===
 
*Describe the cell type for this experiment? 
*What transfection reagent is used for the siRNA tranfection? 
 
NOTE: Transfection reagents are unique and certain cell types respond better to certain cationic lipid (positive charge lipophilic) reagents.
 
*How was transfection efficiency 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.
 
===Cell Confluency===
 
*Adherent  cell confluency?    OR      Suspension cells # used?
 
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: Trying a range of cell confluencies (30, 50, 70%) can reveal an optimal cell density where knockdown is optimal with minimal cell death. Effective confluence can range from 30-80%.
 
===siRNA Concentration===
 
What nanomolar siRNA concentration(s) are tested?  
 
NOTE: Measuring knockdown for a few time points in the 24-72 hour window may indicate the time when RNAi is most optimal. Titrating the siRNA concentration (20-80 nM) for the cells may indicate the best amount to see an effect.
 
Measuring Knockdown
 
How is RNAi measured? Western blot - IF - qPCR - other
 
NOTE: For WB, titrating the antibody may reveal subtle changes in knockdown.
For IF, running secondary controls may indicate nonspecific fluorescence.
 
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.


==References==
==References==
Korey Griffin
2,332

edits

Retrieved from "https://openwetware.org/wiki/Special:MobileDiff/434693"

Navigation menu