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siRNA Illustrated Assay

 This Illustrated Assay Will Cover:

  • A thorough explanation of the theory behind the uses of siRNA.
  • An siRNA protein expression knockdown protocol using pre-made small siRNA.
  • Common problems and variables in both theory and method.

Review: The Central Dogma

  • DNA is transcribed into RNA.
  • RNA is translated into Protein.
  • The tens of thousands of proteins that are created through this process can do everything from creating a new life, to destroying life.

Important Abbreviations

  • siRNA: small, interfering ribonucleic acid
  • mRNA: messenger RNA
  • RISC: RNA induced silencing complex
  • ds: double-stranded
  • nt: nucleotide

siRNA Related Products


The Theory and Methods Behind siRNA

What We Want to Accomplish

  • siRNA keeps proteins from ever being made in the first place by destroying the mRNA that encodes protein.

 

How It Works

  • There are many ways to get siRNA into a cell, which will be covered later.
  • The siRNA, at first, is ds and 20 – 25 nt in length, with 2 nt on each 3’ overhang.

  • The ds siRNA is denatured by helicase protein.

 

 

  • The two siRNA strands, sense and anti-sense, are bound by the RISC related proteins, including at least one endonuclease, and at least one member of the argonaute (eif2C) family.

 

 

 

  • Only the RISC complex containing the anti-sense strand is active, because this is the strand needed to pair with the mRNA.

 

  • The anti-sense RISC binds the mRNA sequence that it is matched perfectly with.  (In this way, only specific proteins can be chosen for ‘silencing’.)

 

  • If just one nt is wrong, the siRNA may not bind.
  • Likewise, as few as 11 contiguous nt matches with an unrelated mRNA can lead to ‘off-target silencing’, which in therapeutics, could be catastrophic.

 

  • If just one nt is wrong, the siRNA may not bind.
  • Likewise, as few as 11 contiguous nt matches with an unrelated mRNA can lead to ‘off-target silencing’, which in therapeutics, could be catastrophic.

  • The active RISC complex is recycled and moves on to destroy more mRNA strands.

 

 

  • With proper siRNA introduction, one protein can be singled out in a cell or organism, and ‘silenced’.

 

 

 

Methods for Introducing siRNA into Cells

Vector Method

  • An expression vector is created that will cause the cell to express the desired siRNA.

 

  • The plasmid expression vector, once in the cell, expresses a small hairpin loop containing the sense and anti-sense siRNA strands.

 

  • The hairpin is cleaved by Dicer, and the sense and anti-sense strands are bound by the RISC complex, and continue the silencing process.

 

Vector Method Comments

  • This method is very good at full silencing because the expression of the siRNA in the cell assures that protein will be fully knocked-out and won’t return.
  • This method is complicated, time consuming and more expensive than other methods, and may not be necessary.

 

Long ds siRNA Method

  • Long ds siRNA (>200 nt) is synthesized and transfected into the cells.

 

  • Once in the cell, long ds siRNA is cleaved multiple times by Dicer, to create the standard siRNA length (20 – 25 nt).  These go on to work as previously described.

 

Long ds siRNA Method, Comments

  • This method may be necessary if you are creating your own siRNA, as long siRNA is much easier and cheaper to produce than short siRNA.
  • siRNA production must be done carefully to assure that Dicer will give the siRNA products desired.
  • ds siRNA sequences >30nt will cause a potent antiviral response by most mammalian cells.

 

Small siRNA Method

  • Synthetic ds siRNA, created in vitro, is transfected into the cells.

 

  • Once present, the ds siRNA is denatured, and bound by the RISC complex, where it goes on to perform its duty.

 

  • Chimeric siRNA is still small ds siRNA.
  • Chimeric siRNA differs in one important way:
  • The strand sequence has been heavily researched and compared to a huge genomic library to assure specific and targeted silencing with guaranteed accuracy.

Small siRNA Method, Comments

  • siRNA in 20-25 nt length is usually purchased directly, and can be purchased in quantities more useful to small labs.
  • Complete knockdown of the gene expression cannot be guaranteed by this method.
  • This method is best for research use only.


Small siRNA Protocol - General Protocols and Recommendations

Small siRNA Protocol

  1. Using healthy cells only, in the tissue culture plate, add antibiotic-free normal growth medium supplemented with FBS (fetal bovine serum).
  2. Incubate for one day at 37°C in a CO2 incubator.
  3. Create or prepare your transfection reagents.
  4. Transfection reagents are normally purchased, and must be specific for your cell line type, and compatible with siRNA.
  5. Sigma Aldrich’s ESCORT line of transfection reagents are a good option.
  6. Follow the transfection reagent makers recommendations for ideal transfection.
  7. Usually, the cells are incubated for 4-8 hours at 37°C in a CO2 incubator, with the transfection reagents containing your siRNA sample.
  8. Next, normal growth medium is added without removing the transfection media, and incubated for an additional day.
  9. After incubation, the medium is aspirated and replaced with fresh normal growth medium.
  10. The cells are then assayed using the desire method within one to three days after the transfection.
  11. After incubation, the medium is aspirated and replaced with fresh normal growth medium.
  12. The cells are then assayed using the desire method within one to three days after the transfection.