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siRNA design (6)


Knock-down of endogenous proteins by RNA interference in HeLa cells new protocol
This doc file is from Anne-Claude Gingras, Sonenberg lab at McGill University. It used synthesized RNAi duplexes to transfect HeLa cells. ...

How to make dsRNA new protocol
This protocol illustrated double sandwiched T7 promoter sequence method (from Kitagawa Group). It is referred to the method described by Kennerdell JR and Carthew RW (1998) with minor modification. ...

Controls for RNAi experiments new protocol
Controls for RNAi experiments powerpoint file (ppt file downloadable). Reference: “Whither RNAi” Nature Cell Biology (2003) vol 5(6): 489-490 ...

A PCR-based strategy for cloning short hairpin sequences: PCR shagging new protocol
The overall approach is to use an RNA polymerase III promoter to drive expression of encoded short hairpin RNA (shRNA). For this purpose we use the U6 snRNA promoter and maintain the transcript initiating G nucleotide of the U6snRNA transcript. There by, hair ...

PCR template method for dsRNA Synthesis (RNAi) new protocol
A method for synthesis of iRNA which allows both RNA strands to be generated simultaneously from a PCR fragment which contains a T7 promoter on each end. (from Carthew/Clemens/Tuschl, MIT) ...

Protocol for siRNA annealing and storage new protocol
This protocol describes how to anneal sense and antisense complementary RNA oligonucleotides to form siRNA and the appropriate storage condition. (Eurogentic) It is crucial to handle RNA oligo under sterile and RNase free conditions. ...

Cloning siRNA duplexes into pSUPER or pSUPERretro vectors new protocol
Strategy and protocol on cloning siRNA duplexes into pSUPER or pSUPERretro vectors for RNAi gene silencing. (Flemington lab at Tulane University). ...

A rapid and sensitive assay for quantification of siRNA efficiency and specificity protocol
Biol. Proced. Online 2005;7:1-7. doi:10.1251/bpo99 Nicola Smart, Peter James Scambler and Paul Richard Riley Molecular Medicine Unit, Institute of Child Health. RNA Interference has rapidly emerged as an efficient procedure for knocking down gene exp ...

RNA Interference of Gene Expression (RNAi) in Cultured Drosophila Cells new protocol
A pdf protocol by Carolyn A.Worby, Nancy Simonson-Leff, and Jack E. Dixon (Science's STKE). ABSTRACT RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented ...

shRNA annealing protocol new protocol
shRNA annealing protocol from Tronolab. ...

Method for PCR-based generation of shRNA libraries from cDNAs new protocol
The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a ...

siRNA experiments new protocol
siRNA experiment guide: 1. Always design 2-4 siRNA sequences for your experiment. An useful online siRNA design tool can be found at Ambion siRNA design tool 2. Chose siRNA with GC content aro ...

Rnai: A Guide to Gene Silencing top rated recommended review
TABLE OF CONTENTS Free sample chaptor supported by biosupplynet.com: Transgene Cosuppression in Animals ...

Oncogene Reviews on RNAi (free access until Dec.24,2004) recommended review
RNAi Issue of Oncogene Reviews - November 1, 2004 (This issue is freely available online until Dec 24, 2004 through the support of Ambion.) Download reviews before the deadline. ...

The siRNA user guide new recommended review
Review on siRNA application written by Tom Tuschl, Sayda Elbashir, Jens Harborth, and Klaus Weber at Tuschl's Lab, Rockefeller University (revised May 6, 2004). ...

RNA Silencing Methods and Protocols review
RNA Silencing Methods and Protocols - by Carmichael, Gordon (University of Connecticut Health Center, Farmington, CT) Human press publication. Link to Amazon.com. ...

Create siRNA Expression Cassettes by PCR new review
Introduction of SECs from Ambion. SECs are PCR products that consist of promoter and terminator sequences flanking a hairpin siRNA template. Upon transfection into cells, the hairpin siRNA is expressed from the PCR product and induces gene specific silencing. ...

RNAi background mini-review by Mary K. Montgomery review
Background RNAi in other systems Mechanism of RNAi Current RNAi studies RNAi protocols from the FireLab at the Carnegie Institution of Washington ...

Optimizing chemical transfection of chemically synthesized siRNA new review
One of the biggest hurdles to obtain reproducible gene silencing results using chemically synthesized siRNAs or DNA constructs that express siRNAs is developing an optimized transfection protocol. The condition for achieving the highest transfection efficiency ...

siRNA Transfection into Primary Neuronal Cells Introduction new review
A hot topic article from GTS inc. Transfection of primary neuronal cells is notoriously difficult. Some advances are given in this short article. ...

A brief introduction of RNA interference review
An introduction of RNA interference and how it was found. (Charles Mallery, Department of Biology, University of Miami) ...

RNAi Protocol Optimization Guide new review
A guidline for RNAi experiments design and protocol optimization. It is brought to you by BD bioscience with RNAi related products. Content: Getting Started * I don’t know where to start * I know which gene I’d like to “knockdown”. Now what? ...

Vector-based siRNA delivery strategies for high-throughput screening of novel target genes new review
An open access review article downloadable in pdf format on vector based siRNA delivery strategies published on Journal of RNAi and Gene Silencing. Journal of RNAi and Gene Silencing (2005), 1(1), 5-11 Meihong Chen, Quan Du, Hong-Yan Zhang, Claes Wahles ...

siRNA transfection new review
Some protocols and overviews on siRNA transfection: Stable Transfection of Mammalian Cells with siRNA expression vectors (Ambion) ...

shRNA short hairpin RNA new review
shRNA shRNA (small hairpin RNA) constructs is an important gene silencing tool for long term and stable gene expression knockdown. Design of shRNA constructs is often very useful for silencing expression of slow turnover genes. There are several commerciall ...

SiRNA and RNAi resources from Ambion site
Ambion's SiRNA and RNAi site hosts many valuable resources and articles including siRNA Database, siRNA Design Tools, RNAi Reference List, siRNA Delivery Resource, miRNA Resource and few reviews. ...

siRNA resources and design tools at CGB site
The CGB database at Karolinska Institute has a number of computational and wet-lab projects utilising siRNA technology. siRNA resources at CGB, Karolinska Institute Introduction Welcome to the unified resource page for siRNA related topics at cgb. The ...

Targeting genes expressed in mammalian cells using siRNAs new recommended troubleshooting
A nature pdf protocol and troubleshooting guide on siRNA. (Thomas Tuschl,Laboratory for RNA Molecular Biology, The Rockefeller University) This is a detailed protocol with troubleshooting solutions in each critical steps. ...

siRNA validated and predicted sequence database new recommended database
Nucleic Acids Res. 2005 January 1; 33(Database Issue): D131–D134. siRNAdb: a database of siRNA sequences Alistair M. Chalk,* Richard E. Warfinge, Patrick Georgii-Hemming, and Erik L. L. Sonnhammer Center for Genomics and Bioinformatics, Karolinska Insti ...

RNA interference - Guid to computational resources new recommended database
A comprehensive resource of RNAi analysis computational tools provided by NCBI. It contains RNAi probe database, terms glossary, high impact review articles, online tools and algorithms for designing RNAi, RNAi project and a list of RNAi technology vendors. ...

siRNA sequence database new database
A siRNA sequence database for published siRNA. This database is NOT complete and is compiled ONLY from published siRNAs--it should only be considered a 'draft' of what such a database could be. (Michael McManus, MIT) ...

Human siRNA Database new database
HuSiDa is a public database that serves as a depository for both, sequences of published functional siRNA molecules targeting human genes and important technical details of the corresponding gene silencing experiments. It aims at supporting the setup and actua ...

Downloadable siRNA DataBase with Thermodynamic & Composition Information database
The database includes siRNA screening experiments reported in the literature. Activity values is expressed as the ratio of the level of a particular mRNA or protein measured in cells after treatment with the experimental siRNA oligonucleotide duplex versus the ...

siRNA sequence list (human, rat, mouse) database
Collections of siRNA sequences from human rat and mouse. (RNA interference) ...

siRNA design tools from Whitehead Institute new recommended software
This site helps you select siRNAs to knock down your gene of interest. After entering your sequence or accession/gi number, choosing pattern for your oligo nucleotides and various filter methods, you will be presented with a list of oligos matching your criter ...

SiRNA design tools from Ambion software
siRNA Target Finder Find siRNA target sites in your mRNA of interest using the published recommendations of Tuschl and colleagues for siRNA design. Once identified, siRNA targets can be sent directly to one of the kit-specific design tools described be ...

siRNA Design Software Using Combination of Online Tools new software
SDS (siRNA Design Software, by TW Lam, Prudence WH Wong, SM Yiu, YC Mui, Vivien Kwan, Department of Computer Science, The University of Hong Kong) is a software tool that helps to design siRNAs for silencing gene expression. The tool takes an mRNA sequence and ...

RNAi technique animation tutorial from Nature new recommended media
This excellent 3D animation is presented by Nature Review Genetics. ...

Gene Silencing and siRNA Technologies (bio.com InFocus series) media
From bio.com InFocus series online broadcasting (mp3 and pdf transcript available). Gene silencing involves cellular processes and mechanisms that result in blockade or inhibition of protein expression at either the transcriptional or translation level. Sma ...

RNAi tutorial: Technologies for RNAi Analysis new media
Technologies for RNAi Analysis: Tools for Researchers from Novice to Advanced (from Biocompare.com). This is a flash animation tutorial also comes with many illustrations. ...

RNAi Analysis: Tools for Researchers from Novice to Advanced new media
The goal of this tutorial is to provide you with a general background and understanding of RNAi technology, introduce you to 3 different RNAi technologies broadly used today, and lead you to vendors and products that can get you started in RNAi research based ...

More rnai protocols

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
presented.

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
silencing.
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
(Forum)
Loss in RNAi reproducibility with increased cell number passages

siRNA transfection
(Forum)
transfection reagents for transfection siRNA

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

siRNA co-transfection
(Forum)
Co-transfection RNAi experiment

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

trouble with siRNA transfection
(Forum)
siRNA Transfection Reagent

FITC-conjugated siRNA
(Forum)
general transfection method to handle with FITC-conjugated siRNA

siRNA oligo transfection
(Forum)
transfect cell with siRNA oligo

pSuper shRNA transfection
(Forum)
get a cell line constantly expressing shRNA

siRNA pool
(Forum)
Mix 3 different siRNAs for transfection


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