How to Work with siRNA

This presentation 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

• Browse all RNAi Products here
• Browse all microRNA Products here

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.

How It Works, II

• The ds siRNA is denatured by helicase protein.

 

 

How It Works, III

• 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.

 

 

 

How It Works, IV

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

 

How It Works, V

• 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’.)

 

How It Works, VI

• 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.

 

How It Works, VII

• 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.

How It Works, VIII

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

 

 

How It Works, IX

• 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.

 

Vector Method, II

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

 

Vector Method, III

• 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.

 

Long ds siRNA Method, II

• 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.

 

Small siRNA Method, II

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

 

Small siRNA Method, III

• 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, I

• Using healthy cells only, in the tissue culture plate, add antibiotic-free normal growth medium supplemented with FBS (fetal bovine serum).
• Incubate for one day at 37°C in a CO2 incubator.

Small siRNA Protocol, II

• Create or prepare your transfection reagents.
• Transfection reagents are normally purchased, and must be specific for your cell line type, and compatible with siRNA.
• Sigma Aldrich’s ESCORT line of transfection reagents are a good option.

Small siRNA Protocol, III

• Follow the transfection reagent makers recommendations for ideal transfection.
• Usually, the cells are incubated for 4-8 hours at 37°C in a CO2 incubator, with the transfection reagents containing your siRNA sample.
• Next, normal growth medium is added without removing the transfection media, and incubated for an additional day.

Small siRNA Protocol, IV

• After incubation, the medium is aspirated and replaced with fresh normal growth medium.
• The cells are then assayed using the desire method within one to three days after the transfection.

Small siRNA Protocol, V

• After incubation, the medium is aspirated and replaced with fresh normal growth medium.
• The cells are then assayed using the desire method within one to three days after the transfection.

 

Questions or comments email technical@novusbio.com