how to analyze a new gene
TRANSCRIPT
How to analyze a new geneA novel gene
Gene expression Gene Functions
Linkage analysis
Domain analysis and structure determination
Protein Location and complex
Biochemical and biological activity
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Protein Expression is regulated at multiple levels
DNA
RNA
Protein
BiologicalActivity
Transcription
Translation
Post-translationalmodification
Gene copy numberChromosome structureMethylation and acetylation
Promoter activityInducible transcription factor
Alternative splicingmRNA transport and stability
Ribosome binding siteCodon usageTermination
Protein folding and targetingProtein stability
PhosphorylationGlycosylationUbiquitinationSumonylationPalmolylation…
Cellular process
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Methods for Analysis of Gene Expression
Analyzing transcription:--- Northern blots--- RNase protection assay--- Reverse transcription PCR--- Real Time PCR--- In situ hybridization--- Primer extention assay
Comparing transcriptomes:--- Differential screening--- Subtractive hybridization--- Differential display--- Array-based methods
Analyzing promoter:--- Nuclear Run-on assay--- DNA Foot-printing assay --- EMSA --- Reporter gene assay--- DNA truncation and
site-directed mutagenesis--- in vitro reconstitution assay--- CHIP assay
Translational analysis:--- Western Blots--- Immunocytochemistry
Immunohistochemistry--- Protein modification--- Proteomics
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Promoter analysis
Western Blot
RT-PCR
Real Time PCR
RNA interference (RNAi)
Topics covered in this lecture
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Promoters: DNA element(s) or sequence which determines the site of transcription initiation of RNA polymerase and may also affect the efficiency of initiation
Promoters are and should be defined functionally as sequences that direct transcription initiation in vivo or in vitro using a functional assay (cause transcription of an RNA).
Promoter analysis
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Mat
rix
Atta
chm
ent r
egio
n
+50-40-500-4000
Regulatory promoter
Gene
Bou
ndar
y el
emen
t
Bou
ndar
y el
emen
t
Mat
rix
Atta
chm
ent r
egio
n
DNA
Enhancer
Core promoter
Components of a typical gene involved in gene regulation
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G/C G/C G/C CGCC T A T A A A Py Py AN T/A Py Py R G A/T C G T G
+1 +30-26-32-38
TATA motif InitiatorDownstream corePromoter element
TF II D
TF II B
TF II A
Polymerase II
Sequence elements in a typical core promoter
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Strategy for promoter analysis
Isolate putative promoter from genomic clone
Sequence putative clone
Determine transcription start site
Establish functional assays
Identify cis-acting DNA elements and trans-acting factors
Determine relevance of these factors
Establish regulatory model of the promoter in vivo and in vitro生物秀-专心做生物
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Nuclear Run-on Transcription Assay
Uninduced cells
Induced cells
Little or no transcription of gene
Active transcription
Lyse cells on ice Pellet nuclei
Add NTPsand
Buffer(One
labeled)
Incubateat 37
for severalminutes
Isolate Radio-labeled
RNA
Hybridize specific radiolabeled RNA to unlabeled cDNA
immobilized on filter
cDN
Afr
omge
ne o
f int
eres
t
Posit
ive
cont
rol
(e.g
β-a
ctin
)
Neg
ativ
e co
ntro
l(e
.g p
lasm
id b
ackb
one)
---An indirect measure of the in vivo rate of transcription initiation from a given gene---The amount of specific radiolabeled RNA observed is approximately proportional to the number of polymerase molecules associated with the gene when the cells were lysed and chilled, which in turn is proportional to the rate of transcription initiation from the gene.
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Primer Extension assay5‘ 3‘ (20-25 bases)
5‘ 3‘
Synthesize primer and label at 5’ end with [γ-32p]ATP and T4
polynucleotide kinase
5‘ 3‘
3‘ 5‘
mRNA
5‘ 3‘
3‘ 5‘
mRNA
Hybridize hot primer to specific mRNA
Extend primer to 5’ end of mRNA using reverse
transcriptase
Analyze radiolabeledDNA on sequencing gel
50-150 bases
G A T C Ext
ende
d pr
imer
Free excess primer
cDNA生物秀-专心做生物
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RNase protection assay
Sp6 promoter
+1
+59-123Hind III
Screen genomic DNA library to find the DNA fragment spanning the region thought to contain the
transcription start site for the gene of interest
Sub-clone Sp6 promoter
+1
+59-123HindIII
Sp6 RNA poltmeraseNTPs (α-32P)UTP
5‘3‘
+1 +59-1235‘3‘
+1 +59-123
mRNA
5‘
3‘
+1 +59
G A T C Undigeste
d probe
RNaseres
isten
t fragm
ent
RNase T1 and/or
RNase A
Hybridize
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5‘ RACE Procedure(Rapid Amplification of cDNA Ends)
AAAAA5‘ 3‘mRNA
AAAAA5‘ 3‘mRNA
5‘3‘
AAAAA5‘ 3‘mRNA
5‘3‘
AAAAA5‘ 3‘mRNA
5‘3‘
5‘3‘
Insert PCR produt into vectorSequence individual clones
Hybridize primer 100-200 bases from 5’ end of mRNA
Reverse Transcription
Ligate oligo of known sequence to 3’ end using RNA ligase orExtend cDNA using terminal transferase(TdT) and dGTP
P C R
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Establishing functional assays for promoter analysis
Transient transfection assay
Stable Transfection assay
In vitro transcription assay
Transgenic assay
Homologous recombination assay
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Transient transfection assay
Reportergene
Reportergene
Promoter of interest
Distant control region of interest
or
Transfect effective and control cell(Inducible genes or cell type specific genes)
Including normalization plasmid
Reportergene
Constitutive promoter(SV40, HSV-TK)
Grow 24-72 hoursStimulation
Harvest cells. Prepare protein extract or mRNA
Measure enzymatic activity of reporter gene product
Measure reporter mRNA levels
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Stable Transfection assay
Promoter of interest
Reportergene
Dominant selectablemarker gene
Constitutive promoter(SV40, HSV-TK)
Transfect cultured cells
Drug selection for 1-4 weeks
Expand several cell clones or pools of drug resistant cells
Harvest cells. Prepare protein extract or mRNA
Mea
sure
enz
ymat
ic a
ctiv
ity
of r
epor
ter
gene
pro
duct
Mea
sure
rep
orte
r m
RN
A le
vels
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In vitro transcription assayPromoter of interest
G-less cassette
A 360-bp sequence devoid of G residues on the noncoding strand.The resulting message is G-less.Transcription should in principle terminate at the end of the G-less cassette.
Incubate plasmid in nuclear extract with ATP, CTP, [32P]UTP
Including 3’-O-Methyl-GTP
Gel electrophoresis analysis
In crude extract, despite extensive dialysis, there are enough contaminating GTP to allow nonspecific RNA synthesis.Acting as chain terminator
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CAT (chloramphenicol acetyltransferase)Transfers radioactive 14C acetyl groups to chloramphenicol.detection by thin layer chromatography and autoradiography
GAL (β-galactosidase)Hydrolyzes colourless galactosides to yield coloured products. Assay change/production of colour
SEAP (secreted human placental alkaline phosphatase)highly-sensitive bioluminescent alkaline phosphatase assay
LUC (luciferase)Oxidizes a luciferin emitting photons. Count photons by luminometer or photon-counting camera. Different luciferases avaiable
GFP (green fluorescent protein)Fluoresces on irradiation with UV – observed upon fluorescence microscopy or measure fluorescence. Use destabilised forms
Choice of Reporter genes
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Common transfection methodsCalcium phosphate:DNA is mixed with calcium chloride in a phosphate buffer, resulting in the formation of a DNA-calcium phosphate precipitate, which is layered on cells and taken up by endocytosis.For both transient and stable transfection of adherent cells
DEAE-dextran:The soluble complex between negatively charged DNA and cationic DEAE-dextran is taken up by endocytosis into cells.For transient transfection of both adherent and non-adherent cells.Rarely useful for stable transfection because of toxicity.
Electroporation:A high voltage electroshock induces or stabilizes pores in the plasma membrane through which the DNA enters the cell.For cells resistant to transfection
Lipofection:Positively charged cationic lipid compoundsExceptionally high efficiency for some cell line
Polycations:
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Dissection of promoter cis-elements (DNA motif mapping)
Identification of cis-elements using in vivo or in vitro protein-DNA interaction methods--- EMSA--- DNase Footprinting
Identification of cis-elements by database analysis--- http://transfac.gbf.de/index.html
Identification of cis-elements by comprehensive mutant analysis--- Deletion analysis--- Generation of internal deletion mutants--- Generation of substitution mutants (Linker scanning)--- Site-directed mutagenesis
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Deletion analysis
Reportergene
Long Promoter of interest
Restriction Enzyme blunt ligation
PCR or
Reporter gene activity生物秀-专心做生物
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Generation of internal deletion mutants by PCR+1
PCR
5‘3‘
5‘3‘
5‘3‘
3‘5‘
3‘5‘
3‘5‘
5‘5‘
3‘3‘
Denature and anneal
5‘5‘
3‘3‘
+1
5‘3‘
3‘5‘
Fill in with Taq and dNTPs
PCR
Clone into reporter plasmids and carry out reporter assay
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Generation of substitution mutants by PCR(Linker scanning)
+1
5‘3‘
3‘5‘
6-10 base pairsSame lengthDifferent sequence
PCR5‘3‘
5‘3‘
3‘5‘
3‘5‘
5‘5‘
3‘3‘
Denature and anneal
5‘5‘
3‘3‘
+1
5‘3‘
3‘5‘
Fill in with Taq and dNTPs
PCR
Clone into reporter plasmids and carry out reporter assay
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Site-directed mutagenesis
Methylated nucleotide
Pfu turbo DNA polymerase
Digest the methylatedparental DNA by DpnI
Transform the circular, nicked dsRNA into
E.coli.. The bacteria will repair the nicks in
the mutated DNA
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Filter Blotting
1979: Towbin et.al Dot blot or Slot blot
ECL detection ofhuman serum transferrin
--- Providing quantitative information about protein expression levels--- Useful for on multiple samples performed in parallel--- Lacking information on protein molecular weight, its isoforms or
post-translational modification
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Western Blotting
SDS-PAGE
Protein mixture
Blot
DetectionECL detection of
human serum transferrin
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PVDF membranes offer better protein retention, physical strength and chemical compatibility
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PVDF is an inherently hydrophobic polymer and will not wet-out in aqueous solutions. In order for a PVDF membrane to be compatiblewith aqueous systems, it must first be wet in a 50% (v/v) or greater concentration of alcohol,methanol,or isopropanol. Complete wetting is evident by a change in the membrane’s appearance from opaque to semi-transparent.
The alcohol is then removed from the membrane by extensive rinsing in water, and the membrane is equilibrated in the appropriate buffer.
Once the membrane is wet, protein binding can be achieved by simply bringing the protein into contact with the membrane.
Pretreatment of PVDF membrane
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Tank Transfer System
Tris/glycine transfer buffer:25 mM Tris base192 mM glycine,10% (v/v) methanolpH 8.3
The methanol added to transfer buffers has two major functions:--- Stabilizes the dimensions of the gel--- Strips complexed SDS from the protein molecules
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1.5 hours 1.5 hours12 hours 12 hours
Electrotransfer Timing: the longer is not always the better
Coomassie Blue Staining
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Long transfer times are best suited for tank systems, which normally require cooling of the unit and internal recirculation of the transfer buffer. In semi-dry transfer, however, prolonged blotting may result in buffer depletion, overheating and gel drying.
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Chromogenic Detection:Coupled enzyme (Alkaline phosphatase, AP) catalyzes a reaction resulting in the deposit of an insoluble colored precipitate.(BCIP: 5-bromo-4-chloro-3-indolylphosphateNBT: nitroblue tetrazolium salt)
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Chemiluminescent Detection:Coupled enzyme(horseradish peroxidase,HRP) catalyzes a reaction that results in the production of visible light.
Fluorescent Detection:
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cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysateby extraction with phenol:chloroform at reduced pH. Many samples can be processed simultaneously and speedily. The yield of total RNA depends on the tissue or cell resource and is generally in the range of 4-7 µg/ml starting tissue or 5-10 µg/106 cells.
Prepare all reagents with DEPC-treated H2O.
Guanidnium thiocyanate is a strong denaturing agent, preventing RNA degradation
Proteinase K degrades protein contaimination
RNAase-free DNAase degrades DNA.
Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-chloroformExtraction
Phenol layer
Protein precipitate
Aqueous layer(DNA and RNA)
Neutral pH
Phenol layer(DNA)
Protein precipitate
Aqueous layer( RNA)
Acidic pH
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Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography
Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian cells. A combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA.
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Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase
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The Evolution of PCR to Real-Time PCR
PCR has completely revolutionized the detection of RNA and DNA. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring.
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Poor Precision
Low sensitivity
Short dynamic range < 2 logs
Low resolution
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not very quantitative
Post PCR processing
Limitations of Traditional PCR
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What is Real-Time PCR
A PCR system in which we can monitor the amplification reaction as it is occuring. It incorporates the ability to directly measure and quantify the reaction while amplification is taking place.
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A basic PCR run can be broken up into three phases:
Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.
Linear (High Variability): The reaction components arebeing consumed, the reaction is slowing, and products are starting to degrade.
Plateau : The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
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threshold The Threshold line is the level ofdetection or the point at which a reaction reaches a fluorescent intensityabove background. The threshold line is set in the exponential phase ofthe amplification for the most accurate reading. The cycle at which thesample reaches this level is called the Cycle Threshold, Ct.
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--- Glyceraldehyde-3-phosphate dehydrogenase mRNA--- Beta-actin mRNA--- MHC I (major histocompatability complex I) mRNA--- Cyclophilin mRNA--- mRNAs for certain ribosomal proteins RPLP0
28S or 18S rRNA
Common Standards for Real Time PCR
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REAL TIME PCR
• kinetic approach• early stages• while still linear
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2a. excitation filters
2b. emission filters
1. halogen tungsten lamp
4. sample plate
3. intensifier 5. ccd
detector 350,000 pixels
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The 5’ exo-nuclease activity of AmpliTaq® Polymerase and FRET (Fluorescent Resonant Energy Transfer) makes it possible to detect PCR amplification in Real-Time.
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The Scorpions uni-probe consists of a single-stranded bi-labeled fluorescent probe sequence held in a hairpin-loop conformation (approx. 20 to 25 nt) by complementary stem sequences (approx. 4 to 6 nt) on both ends of the probe. The probe contains a 5’end reporter dye and an internal quencher dye directly linked to the 5’end of a PCR primer via a blocker. The blocker prevents Taq DNA polymerase from extending the PCR primer. The close proximity of the reporter dye to the quencher dye causes the quenching of the reporter's natural fluorescence. At the beginning of the real-time quantitative PCR reaction, Taq DNA polymerase extends the PCR primer and synthesizes the complementary strand of the specific target sequence. During the next cycle, the hairpin-loop unfolds and the loop- region of the probe hybridizes intramolecularly to the newly-synthesized target sequence. The reporter is excited by light from the real-time quantitative PCR instrument (hg ). Now that the reporter dye is no longer in close proximity to the 1 quencher dye, fluorescence emission may take place (hg ).
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SYBR Green chemistry is an alternate method used to perform real-time PCR analysis. SYBR Green is a dye that binds the Minor Groove of double stranded DNA. When SYBR Green dye binds to double stranded DNA, the intensity of the fluorescent emissions increases. As moredouble stranded amplicons are produced, SYBR Green dye signal will increase.
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RNA interference (RNAi)post-transcriptional gene silencing (PTGS)
Antisense-mediated silencing: large amounts of a nucleic acid whose sequence is complementary to the target messenger RNA are delivered into the cytoplasm of a cell. Base pairing between the 'sense' mRNA sequence and the complementary 'antisense' interfering nucleic acid is thought to passively block the processing or translation of mRNA, or result in the recruitment of nucleases that promote mRNA destruction.Unexpected results:---Both the antisense and the control sense RNA preparations induced silencing of C. elegans par-1 mRNA to block par-1 expression.---Silencing effect could be transmitted in the germ line.---The silencing effect could also spread from tissue to tissue within the injected animal.Hypothesis:---The properties of RNAi demands the existence of cellular mechanisms that initiate and amplify the silencing signal, and suggest that the RNAi mechanism represents an active organismal response to foreign RNA.---dsRNA, often encountered by cells during viral infection, might itself be the initial trigger. dsRNA proved to be an extremely potent activator of RNAi — at least 10-fold and perhaps 100-fold more effective than purified preparations of single-stranded RNA.
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Natural mechanism of RNA interference
Dicer: RNase III enzymeRISC: RNA-induced silencing complex
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Advantages and Disadvantages of Different Gene Suppression Strategies
Laborious to produceExpensive
Lethal mutants may prevent embryonic development
100% gene silencing“Knockout mouse”
Often nonspecificMay not be availableEase of administrationSmall molecule
inhibitors
Transfection-dependentUnanticipated side effects
Ability to determine functions ofdiscrete regions of a proteinStable suppression possible
dominant negativemutant gene
Variable efficacy and specificityTransfection-dependentSimple, inexpensiveAntisense DNA
oligonucleotides
Transfection-dependent“Knockdown” not
“knockout”
Potent, specificRNAi
DisadvantageAdvantage
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siRNA design
There are expanding libraries of validated, commercially available siRNAs directed toward some commonly targeted genes. These may be of use, if available. However, if the gene of interest has not been targeted using siRNAbefore, a novel siRNA must be developed.
Selection of the targeted region is currently a trial-and-error process, but with the likelihood of 80–90% success , given a large enough random selection of target genes
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Methods to generate short RNAsthat silence gene expression
Silencing by RNAs that are generated in vitro
Silencing by RNAs that are generated in vivo
(H1 and U6)
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Advantages and Disadvantages of Different siRNA Delivery Strategies
More labor intensive to generate
Potential biohazard
May be effective in cells resistant to transfection with
dsRNA and plasmidsIntegration produces stable
RNAi even in theabsence of a selection
pressure
Virus-mediated
More labor intensive to generate
Transfection-dependent
More economical for multiple sequences
Stable RNAi achievable using selection marker
DNA plasmid vector or cassette
Transient RNAiexpensive for multiple siRNA
Rapid synthesisHigh purity using chemical
synthesis
Chemical and in vitro enzymatic synthesis
DisadvantageAdvantage
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Selection of siRNA duplexes
[1]Select the target region from the open reading frame of a given cDNA sequence preferably 50 to 100 nt downstream of the start codon. Avoid 5′ or 3′ untranslated regions (UTRs) or regions close to the start codon as these may be richer in regulatory protein binding sites. [2]Search for sequences 5′-AA(N19)UU . Choose those with approximately 50% G/C content ( from 32 to 79% G/C content ). If not, follow (B).[3] Blast-search the selected siRNA sequences against EST libraries or mRNA sequences of the respective organism to ensure that only a single gene is targeted.[4] It may be advisable to synthesize several siRNA duplexes to check the silencing effectivity. A nonspecific siRNA duplex is needed as control.
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Preparation of siRNA duplexes by Chemical Synthesis
Proligo (Hamburg, Germany, www.proligo.com)Dharmacon Research (Lafayette, CO, www.dharmacon.com)Pierce Chemical (www.perbio.com)Glen Research (Sterling, VA, www.glenres.com)ChemGenes (Ashland, MA, www.chemgenes.com)Cruachem (Glasgow, UK, www.cruachem.com).
Analysis of siRNA duplex formation
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Schematic for a typical RNAi experiment using chemically synthesized siRNA
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Silencing of a green fluorescent protein (GFP) reporter in C. elegans
RNAi Control
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Longer exposure
A B
Silencing of the cytoskeletalintermediate filament protein vimentin
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Silencing of SV40 large T antigen in a stably transformed rat fibroblast cell line
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