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siRNA design (6)

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Overview of RNA interference and related processes.
(Curr Protoc Mol Biol. 2003 May;Chapter 26:Unit 26.1.)
Baulcombe D.

The Sainsbury Laboratory John Innes Center, Norwich, United Kingdom.

The history of RNA interference (RNAi) has unfolded rapidly since 1997 with a
series of discoveries from plants, fungi, and animals. Initially, the interest
was directed towards development of gene silencing technology that could be
applied in research, medical therapy, and crop improvement. However, as the
underlying mechanism was revealed, it became apparent that RNAi manifests a novel
system of genetic regulation that was only hinted at by previous data. This
overview unit gives a brief history of the field, describes the natural role of
RNAi and related processes, identifies strategies for RNAi and cosuppression in
plants and animals, and describes methods for detection and characterization of
siRNA and miRNA.

RNA interference in cultured Drosophila cells.
(Curr Protoc Mol Biol. 2004 Feb;Chapter 26:Unit 26.5.)
Worby CA, Dixon JE.

University of California at San Diego, La Jolla, California, USA.

RNA interference (RNAi) can be used to silence genes in a number of species,
including plants, nematodes, protozoans, Drosophila melanogaster, mouse embryos,
and mammalian and Drosophila cell cultures. Drosophila cell culture provides the
opportunity to study signal transduction pathways and protein function in a
simple, well defined cell culture paradigm. Furthermore, because Drosophila are
RNAi responsive, the results obtained from experiments performed on cultured
cells can be confirmed in the whole organism. RNAi takes advantage of the unique
ability of double-stranded RNA (dsRNA) molecules to induce gene silencing in a
highly specific manner. This phenomenon is efficacious and long lived, being
passed to subsequent generations in Drosophila cell culture. To date, many
Drosophila cell lines tested respond to dsRNAs by ablating expression of the
target protein. Furthermore, all dsRNAs tested have been efficacious at silencing
the target gene. Drosophila cell cultures are simple, easily manipulated model
systems that will facilitate loss of function studies applicable to a wide
variety of questions.

Gene silencing by RNAi in mammalian cells.
(Curr Protoc Mol Biol. 2003 May;Chapter 26:Unit 26.2.)
John M, Geick A, Hadwiger P, Vornlocher HP, Heidenreich O.

Ribopharma AG, Kulmbach, Germany.

This unit provides information how to use short interfering RNA (siRNA) for
sequence specific gene silencing in mammalian cells. Several ways for siRNA
generation and optimisation, as well as recommendations for cell transfection are

Cloning of small RNA molecules.
(Curr Protoc Mol Biol. 2005 Nov;Chapter 26:Unit 26.4.)
Pfeffer S, Lagos-Quintana M, Tuschl T.

The Rockefeller University, New York, New York, USA.

Small RNAs that are derived from dsRNA precursors act as guide RNAs during
sequence-specific epigenetic regulation of eukaryotic gene expression. These
small regulatory RNAs are between 20 and 30 nucleotides in length, and fall into
one or more of the following categories: small interfering RNAs (siRNAs),
microRNAs (miRNAs), and heterochromatic siRNAs (hsiRNAs). Procedures to record
the profile of small RNAs expressed in cultured cells or tissues are described.
The small RNAs are directionally cloned after isolation from total RNA. The
methods rely on T4 RNA ligase-based joining of adapter oligonucleotides to the 3'
and 5' termini of the pool of small RNAs. The ligation products are reverse
transcribed and PCR-amplified. It is recommended to directionally concatamerize
the relatively short PCR products before cloning in order to increase the number
of RNA sequences obtained per clone.

(Selected reviews on siRNA techniques and applications)
1: De Paula D, Bentley MV, Mahato RI.
Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting.
RNA. 2007 Feb 28; [Epub ahead of print]

2: Patzel V.
In silico selection of active siRNA.
Drug Discov Today. 2007 Feb;12(3-4):139-48. Epub 2006 Dec 13.

3: Crombez L, Charnet A, Morris MC, Aldrian-Herrada G, Heitz F, Divita G.
A non-covalent peptide-based strategy for siRNA delivery.
Biochem Soc Trans. 2007 Feb;35(Pt 1):44-6.

4: Fuchs U, Borkhardt A.
The application of siRNA technology to cancer biology discovery.
Adv Cancer Res. 2007;96:75-102. Review.

5: Inoue A, Sawata SY, Taira K.
Molecular design and delivery of siRNA.
J Drug Target. 2006;14(7):448-55. Review.

6: Kurreck J.
siRNA Efficiency: Structure or Sequence-That Is the Question.
J Biomed Biotechnol. 2006;2006(4):83757.

7: Zhuang F, Liu YH.
Usefulness of the luciferase reporter system to test the efficacy of siRNA.
Methods Mol Biol. 2006;342:181-7. Review.

8: Toub N, Malvy C, Fattal E, Couvreur P.
Innovative nanotechnologies for the delivery of oligonucleotides and siRNA.
Biomed Pharmacother. 2006 Nov;60(9):607-20. Epub 2006 Aug 31. Review.

9: Ikeda Y, Taira K.
Ligand-targeted delivery of therapeutic siRNA.
Pharm Res. 2006 Aug;23(8):1631-40. Review.

10: Zhang HY, Du Q, Wahlestedt C, Liang Z.
RNA Interference with chemically modified siRNA.
Curr Top Med Chem. 2006;6(9):893-900. Review.

11: Gilmore IR, Fox SP, Hollins AJ, Akhtar S.
Delivery strategies for siRNA-mediated gene silencing.
Curr Drug Deliv. 2006 Apr;3(2):147-5. Review.

12: Morris KV.
siRNA-mediated transcriptional gene silencing: the potential mechanism and a
possible role in the histone code.
Cell Mol Life Sci. 2005 Dec;62(24):3057-66. Review.

13: Amarzguioui M, Rossi JJ, Kim D.
Approaches for chemically synthesized siRNA and vector-mediated RNAi.
FEBS Lett. 2005 Oct 31;579(26):5974-81. Epub 2005 Sep 20. Review.

14: Vaucheret H.
MicroRNA-dependent trans-acting siRNA production.
Sci STKE. 2005 Sep 6;2005(300):pe43. Review.

15: Akashi H, Matsumoto S, Taira K.
Gene discovery by ribozyme and siRNA libraries.
Nat Rev Mol Cell Biol. 2005 May;6(5):413-22. Review.

16: Juliano RL.
Peptide-oligonucleotide conjugates for the delivery of antisense and siRNA.
Curr Opin Mol Ther. 2005 Apr;7(2):132-6. Review.

17: Kawasaki H, Taira K, Morris KV.
siRNA induced transcriptional gene silencing in mammalian cells.
Cell Cycle. 2005 Mar;4(3):442-8. Epub 2005 Mar 7. Review.

18: Gilmore IR, Fox SP, Hollins AJ, Sohail M, Akhtar S.
The design and exogenous delivery of siRNA for post-transcriptional gene
J Drug Target. 2004 Jul;12(6):315-40. Review.

19: Scanlon KJ.
Anti-genes: siRNA, ribozymes and antisense.
Curr Pharm Biotechnol. 2004 Oct;5(5):415-20. Review.

20: Sachse C, Echeverri CJ.
Oncology studies using siRNA libraries: the dawn of RNAi-based genomics.
Oncogene. 2004 Nov 1;23(51):8384-91. Review.

(siRNA transfection)
siRNA transfection efficiency is dependent on a variety of factors. To chose the right siRNA transfection reagent is the key to achieve maximum silencing effect of siRNA. The most appropriate way to measure the silencing effect is through protein expression detection of the targeted gene. Several siRNA transfection reagents are available from different vendors to deliver siRNA duplex into cells. The general guideline is to chose siRNA transfection reagents with maximum transfection efficiency and minimum toxicity.

Recommended siRNA user guide:

Selection of siRNA duplexes from the target mRNA sequence (Tuschl lab, Rockefeller University)

RNA interference books
(Biowww bookshelf)
RNAi gene silencing techniques

Cell passage number and RNAi efficacy
Loss in RNAi reproducibility with increased cell number passages

siRNA transfection
transfection reagents for transfection siRNA

Troubles to transfect siRNA
transfecting siRNA of Sp1 into MCF-7 cells

siRNA co-transfection
Co-transfection RNAi experiment

siRNA - augmentaion instead of knockdown
knockdown Luciferase gene using luciferase reporter plasmid in HEK293 cells and Oligonucleotide Luciferase

trouble with siRNA transfection
siRNA Transfection Reagent

FITC-conjugated siRNA
general transfection method to handle with FITC-conjugated siRNA

siRNA oligo transfection
transfect cell with siRNA oligo

pSuper shRNA transfection
get a cell line constantly expressing shRNA

siRNA pool
Mix 3 different siRNAs for transfection

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