siRNA

1siRNA
Overview and Technical Tips

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siRNA

2CONTENTS
3–4

Introduction

5–7

Applications

8–10

How Does It Work?

11–13

Handy Tips

14–18

Troubleshooting

19

Conclusions

20

Further References

21

Contact Us

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siRNA3

INTRODUCTION
At A Glance

In Conclusion–– Applications as potential therapeutics and in drug development.

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INTRODUCTION
At A Glance

RNAi interference (RNAi):–– RNAi is a post-transcriptional gene 		silencing process.
–– RNAi leads to sequence specific 		
degradation of mRNA.
–– Highly specific process.
–– Important genome regulationand defence mechanism.

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siRNA

5APPLICATIONSGeneral IdeassiRNAs:–– Small interfering RNAs.
–– 21–25 nt long fragments.
–– Bind to complementary target mRNA.
Advantages of gene silencing:–– Cost effective method.
–– Fast design of siRNA, mRNA sequenceis needed.
–– Highly specific method.
–– Downregulation of target genes.
–– Helpful tool for the analysis of genes
and their functions.
–– Powerful tool in gene therapy.
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siRNA

6APPLICATIONSBig Potential
From Small RNAs

Powerful tool for functional genomics:–– An accurate and potent genesilencing method.

"Billion Dollar Breakthrough"- Fortune–– Knock-downs are quite easy.
–– Study of functions.
–– Variety of controls.

"Technology Of The Year"- Science (2002)–– A lot of published data exist ofsiRNA targeting specific genes.
In Conclusion–– Small RNAs have been identified to play multiple biological roles.
–– Currently the most widely used gene-silencing technique.

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siRNA7APPLICATIONS
Oncology–– Analysis of signaling molecules.
–– Defining oncogenes.

Infectious Diseases–– Virus targeting:Some siRNA candiates
entered clinical trials only 10years after the discovery of theRNAi mechanism.

HIVHepatitis
Respiratory infections
Stem Cells–– Observation of tumor phenotype.

Hematology–– Designing disease models (hematologicdisorders are loss of function diseases).

Other Diseases–– Huntington, Macular Degeneration.

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siRNA8HOW DOES IT WORK?
1.

1. dsRNA is processed into sense/antisence
RNA by Dicer.

2.

2. siRNA associates with RISC.

3.

3. Antisense siRNA/RISC complex associateswith the target mRNA.

4.

4. RISC cuts the target mRNA.

5.

5. mRNA degradation.

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siRNA

9HOW DOES IT WORK?
Inspired by siRNA

siRNA sequences can be tailoredto block disease-causing genes.

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HOW DOES IT WORK?
Protocol Overview

Important factors influencing
siRNA experiments
Cell type
Cell growth rate
Cell density
Cell viability
Transfection method
Quality/quantity of siRNATime of transfectionwww.ptglab.com

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HANDY TIPS
RNase-freeEnvironment–– Use RNase-free tips.
–– Use pipettes only for RNA work.
–– Use gloves.
–– Changes gloves after touching any surface.
–– Clean your work surface with a RNase-decontaminating
solution or wipes.
–– Working in a RNase free zone, no air vents.

Working With A
New Target/siRNA/
Cell Type–– Requires multiple test transfections to optimize thebest conditions.
–– Transfection efficiency should be as high as possible.
–– Fluorescently labeled siRNA simplifies targeting of theknock-down effect.
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HANDY TIPSValidation OfsiRNA Data–– Use positive/negative controls.
–– Use a second siRNA against the same target.
–– Titrate the siRNA concentration.
–– Monitor RNA and protein level.

Optimizing siRNA
Delivery Into
Cultured Cells–– Keep culture conditions consistent.
–– Healthy cells.
–– Optimal cell density.
–– Optimize culture conditions (media, etc).
–– Optimize transfection reagent.

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HANDY TIPS
Designing siRNA–– Around 21–23nt long.
–– G/C content: 30–50%.
–– No basepair mismatch.
–– siRNA should not bind to introns.
–– No sequence that shows homology other coding
sequences (BLAST).
–– Work with two or three different siRNA constructs to getreliable results.

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TROUBLESHOOTING
FAQs
FAQs

Recommendations

Serum or serum-free medium?

Transfection reagents require serum-free medium for dilution of thesiRNA complex.
Serum quality/lot might affect the experiment.

Antibiotics orantibiotics-free medium?

Dependent on combination of cell type and transfection reagent:
Cell permeability is very sensitive during transfection. Antibiotics
can cause cell death.

Replacement oftransfection medium?

In general it can be replaced after 6h as at this time the transfection will becompleted. Not required step, depends on tranfection reagent.

Storage of siRNA reagents?

siRNA: frozen (clean tube before use), reagents: 4°C.

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TROUBLESHOOTING
FAQs
FAQs

Recommendations

siRNA concentration?

The lowest functional siRNA amount evaluated in test transfection should
be used for the experiments.

Duration of siRNA silencing?

In general, the silencing effect can be observed earliest after 24h. It retains
cell type dependent for 4–7 days.

Stock solution of siRNA.

Consult manufacturer’s instructions, generally between 50–100 µM.

Working solution of siRNA?

Has to be optimized. Generally, a rage of 5–100 nM is used.

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TROUBLESHOOTINGControls
Control type

Recommendations

Read-out

Analyse mRNA and protein level.

Use lowest effective
siRNA concentration

Higher concentrations of siRNA lead to more off-target effects.

GAPDH siRNA control

GAPDH is high expressed in virtually all mammalian cells. It is a
useful tool for evaluating transfection efficiency and cell viability.

Transfection of two or moredifferent siRNA against thetarget of interest

Helps to identify sequence specific off-target effects.

New target/siRNA/cell type

Requires multiple test transfections to optimize the best conditions.

Toxic impact

Cell sample just treated with the transfection reagent.

Endogenous protein level

Non-treated/transfected cell sample.

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TROUBLESHOOTING
Controls
Control type

Recommendations

Titrate siRNA amountUse different concentrations.

siRNA conjugated to afluorescence label

Microscopic evaluation of transfection efficiency.

Include a cell line with a known
high transfection efficiency.

If the cell line of interest is difficult to transfect, use a control cell line.

Scrambled siRNA

Helps to differentiate between sequence-specific silencing from non-specificsilencing affects.

Time points

Check silencing affects at different post-transfection time points.

Recombinant protein

Re-introduce the protein by transfection with the recombinant protein.

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TROUBLESHOOTING
Unhealthy Cells
Recommendations
Passage cell lines frequently, do not allow cells to overgrown.
Passage fresh cells lines at least 1–2 times before the experiment.
Usually a high cell density is needed > 70 %*.
Transfect cells at the same passage numbers.
* Cell density and passage number depends on the type of cell line.

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CONCLUSIONS–– RNAi is an important part of the cellular machinery thatprovides viral immunity and mechanism for the controlof gene expression.
–– siRNA triggers function in the RNAi mechanism result ingene suppression.
–– Potential therapeutic application include viruses and cancer.
–– Technical challenges:
Stability
Targeting
Off-target effects
Immune response
Delivery–– The field is relatively young and much remains to be discovered.

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FURTHER REFERENCES–– K. Huppi, S. Martin, et al., Defining and Assaying RNAi in Mammalian Cells. Mol. Cell.
17(1), 1-10 (2005).
–– Whither RNAi? Nat. Cell. Biol. 5(6), 489-90 (2003).
–– L.P. Lim, N.C. Lau, et al., Microarray analysis shows that some microRNAs down regulate
large numbers of target mRNAs. Nature. 433(7027), 769-773 (2005).
–– F. Barthel et al, Gene transfer optimization with lipospermine-coated DNA, DNA Cell
Biol. 12, 553–60( 1993).
–– P.L. Felgner et al, Enhanced gene delivery and mechanism studies with a novel seriesof cationic lipid formulations. J. Biol. Chem. 269, 2550–61 (1994).
–– D.H. Kim et al., Nature Reviews Genetics 8, 173–184 (2007).
–– S. Loyter et al., Mechanisms of DNA uptake by mammalian cells: Fate of exogenouslyadded DNA monitored by the use of fluorescent dyes. Proc. Natl. Acad. Sci. USA 79,422–6. (1982).
–– D.S . Anson et al., The use of retroviral vectors for gene therapy-what are the risks?
A review of retroviral pathogenesis and its relevance to retroviral vector-mediatedgene delivery. Genet. Vaccines Ther. 2, 9. (2004).

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siRNA21

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United Kingdom

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China Officeservice@ptglab.com

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Please visit us at www.ptglab.com formore information about our antibodies
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