key: cord-0797707-f18q7d4q
authors: Tsai, Wen-Hui; Chang, Wen-Tsan
title: Construction of Simple and Efficient siRNA Validation Systems for Screening and Identification of Effective RNAi-Targeted Sequences from Mammalian Genes
date: 2013-08-13
journal: Gene Function Analysis
DOI: 10.1007/978-1-62703-721-1_15
sha: ebd0a7f4271d53155a827a93603ca5fe51f29c5a
doc_id: 797707
cord_uid: f18q7d4q

RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene silencing induced by double-stranded RNAs (dsRNAs) that mediate sequence-specifi c cleavage of the cognate RNA transcripts and in turn trigger complete degradation of the disruptive RNA fragments, resulting in reduction or loss of gene activities [ 1 , 2 ] . During the processes of RNAi-mediated gene silencing, the dsRNAs are fi rst recognized and cleaved into 21-to 23-nucleotide (nt) small interfering RNA (siRNA) duplexes with symmetrical 2-nt 3′ overhangs by dsRNA-specifi c RNase IIIrelated endonuclease, Dicer [ 3 , 4 ] . The produced siRNAs are efficiently incorporated into RNA-induced silencing complex (RISC) to form a ribonucleoprotein complex that fi rst unwinds the siRNA duplexes and selectively degrades the sense strand of siRNA. The single antisense siRNA-coupled RISC is afterwards guided to catalyze the endonucleolytic cleavage of homologous RNA transcripts at the site where the antisense siRNAs is complementarily bound [ 5 , 6 ] . Subsequently, the resulting cleaved RNA fragments are immediately subjected to exonucleolytic destruction by the action of exoribonuclease.

Originally, RNAi was identifi ed as involved in the response to exogenous pathogenic and endogenous parasitic nucleic acids [ 7 , 8 ] and also as participating in the basic cellular functions, including gene regulation and heterochromatin formation [ 9 -11 ] . Recently, it has become an extremely powerful study tool for reverse functional genomics [ 12 -15 ] and a remarkably potent strategy for gene silencing-based therapeutics [ 16 , 17 ] . Compared to other gene silencing reagents, such as antisense oligonucleotides (ODNs), ribozymes, and DNAzymes, dsRNAs including siRNAs and short hairpin RNAs (shRNAs) have apparently become the most powerful and widely used gene silencing reagents for manipulating gene activity in mammalians [ 18 ] . There are mainly two approaches in generating active dsRNAs in mammalian cells by exogenous delivery of synthetic siRNAs [ 19 , 20 ] or shRNAs [ 21 ] and endogenous vector-expressed shRNAs [ 22 -25 ] or siRNAs formed by annealing two complementary sense and antisense RNAs [ 26 -28 ] .

A large number of studies have shown that not all of the RNAi-targeted sequences selected from a target gene exhibit the same effi ciency in inhibiting gene expression. Practically, only a small number of siRNAs are capable of inducing highly effective gene silencing in a sequence-specifi c manner [ 29 ] . Moreover, the silencing effi ciency of siRNAs is dependent on the specifi city of the target sequences within a gene and can only be measured experimentally based on the inhibition of the target gene expression. In order to select functional and effective siRNAs, it is necessary to design, synthesize, and screen many distinct siRNAs, which is expensive due to the cost of chemical synthesis of RNA oligonucleotides. Several previous studies have suggested that the secondary structures of mRNA and mRNA-binding proteins might interfere with the target site accessibility for RISC; therefore, the rational design strategy for selecting effective siRNAs is not fully programmable [ 30 -33 ] . In addition, extensive large-scale and systematic analyses of the siRNA-specifi c features revealed that siRNA might have sequence-specifi c characteristics associated with its functionality [ 29 , 34 -37 ] . Thus, these studies indicate that the effi cacy of siRNA is not totally secondary structure dependent and strongly suggest that the sequence properties of siRNA may play the major and most important role in determining inhibition effi ciency.

The key challenge in achieving highly effective gene silencing, particularly for the purpose of the therapeutics, is mainly dependent on the effectiveness and specifi city of the RNAi-targeted sequence. Previously, the effective siRNAs are identifi ed on the basis of their abilities to inhibit the expression of cognate sequences in an ectopically expressed target gene-reporter fusion chimeric mRNA [ 38 ] . The described target gene-reporter-based siRNA validation system totally depends on the availability of cDNA clones, and this may limit the high-throughput application of the method. Besides, the chimeric mRNA of target genereporter fusion construct may encode an impaired fusion protein that exhibits a low reporter activity, which interferes with the screening and identifi cation of effective siRNAs. Recently, a reporter-based siRNA validation system has been published in which the validating system is constructed by fusing a short synthetic DNA fragment containing an RNAi-targeted sequence, instead of cDNA, with a reporter gene [ 39 , 40 ] . However, to generate the corresponding triggering siRNA, it is necessary to have either a synthetic siRNA (or shRNA) or another synthetic DNA fragment for constructing siRNA (or shRNA) expression vector. Thus, this system is ineffi cient and cost intensive, especially for large-scale studies.

To facilitate large-scale functional genomics in mammals, especially for novel genes, it is important to have a rational design strategy for selecting potentially effective siRNAs and measure the effi cacy of the designed siRNAs by a simple and fully robust validating system. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been developed in our laboratory for functional screening and identifi cation of effective RNAi-targeted sequences in mammalian genes [ 41 ] . In this system, only a short synthetic DNA fragment is needed to construct both the targeting reporter and triggering siRNA expression vectors. Because only a readily available short synthetic DNA fragment is needed to construct both the targeting reporter and triggering siRNA expression vectors, the protocols described in this chapter provide a novel system that not only greatly facilitates large-scale loss-of-function genetic screens in mammalian cells but also provides the basis for an improved approach to screen and identify the most potent siRNA for therapeutic purposes. 13. TE: 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA; stored at room temperature.

The methods described in this section outline (1) the strategy and experimental design for screening and identifying effective RNAitargeted sequences, (2) the molecular characteristics of designed and selected RNAi-targeted sequences, (3) the construction of

reporter-based siRNA validation system including targeting reporter and triggering siRNA expression vectors, (4) the functional assessment of designed and selected RNAi-targeted sequences in this reporter-based siRNA validation system, and (5) the application of this reporter-based siRNA validation system to identify highly effective novel siRNAs.

To screen and identify the effective RNAi-targeted sequences more robustly and cost effectively, we have established a reliable and quantitative reporter-based siRNA validation system requiring only a short synthetic DNA fragment [ 41 ] . This system is composed of a short synthetic DNA fragment and two expression vectors for targeting reporter and triggering siRNA expressions ( see Fig. 1 ). The short synthetic DNA fragment is generated by annealing two complementary sense-and antisense-oligonucleotides that contain a unique RNAi-targeted sequence with 19 nt in length fl anked by fi ve consecutive adenosine and thymidine residues (As and Ts) at the 5′ and 3′ ends as an effi cient termination signal for transcription of the antisense and sense RNA, respectively ( see Fig. 1b ).

The unique RNAi-targeted sequence is selected and designed from protein-coding region or 3′-untranslated region (3′-UTR) of the target gene according to the sequence-specifi c characteristics of the effective siRNAs as described briefl y in Subheading 1 and thoroughly in Subheading 3.2 . To construct the targeting reporter vector, a short synthetic DNA fragment containing a unique RNAitargeted sequence of interest is fused with a reporter gene at the 5′-and 3′-UTR or is inserted within the reporter gene without interfering in its activity ( see Fig. 1a ). To make the greatest value and utility of this short synthetic DNA fragment, simultaneously, it is also cloned into a specifi c triggering siRNA expression vector that contains two functional convergent RNA Pol III promoters ( see Fig. 1c ; Note 2 ).

Effi cacy of the siRNAs is measured by their abilities to inhibit expression of the targeted reporter gene, which contains the corresponding short synthetic DNA fragment, with easily quantifi ed readouts including EGFP or Fluc. In addition, to make this system complete, it is convenient to include an excellent in vitro cell model, which not only provides an easy cell culture system but also has high transfection effi ciency. Because this system is used for screening the highly effective RNAi-targeted sequences directly against genes with a wide range of biological functions, it is better to perform the screening in a nonhuman and non-mouse cell line. To fi t these criteria, the baby hamster kidney fi broblast BHK is chosen as an in vitro experimental model. In addition, the experimental procedures including cell culture conditions and transfection protocols have been optimized and standardized.

Strategy and experimental design for screening effective RNAi-targeted sequences using reporter-based siRNA validation system. This reporter-based siRNA validation system is composed of a short synthetic DNA fragment ( b ) and two expression vectors for targeting reporter ( a ) and triggering siRNA ( c ) expression. The short synthetic DNA fragment contains a unique RNAi-targeted sequence with 19 nt in length and two specifi c restriction enzyme-compatible ends Hin dIII and Bgl II for cloning into the Hin dIII/ Bgl II-digested targeting reporter and triggering siRNA expression vectors simultaneously. The restriction enzymecompatible ends of Hin dIII (5′-AGCT ) and Bgl II (5′-GATC ) are underlined . The targeting reporter vector not only contains the reporter gene expression cassette driven by RNA Pol II promoter but also includes two unique restriction enzyme sites, Hin dIII (H) and Bgl II (B), for cloning of the RNAi-targeted sequence at the 5′-or 3′-UTR or insertion within the reporter gene without disrupting its activity. The positions for inserting the RNAi-targeted sequence are marked with black triangles . The triggering siRNA expression vector contains two convergent RNA Pol III promoters to drive the expression of both the sense and antisense strands of siRNA, respectively. The short synthetic DNA fragment is also cloned into the Hin dIII (H) and Bgl II (B) sites in triggering siRNA expression vector

The effi ciency of RNAi-induced gene silencing is mainly dependent on the effectiveness and specifi city of the RNAi-targeted sequences. To gain the functionally effective siRNAs, it is necessary to design, synthesize, and screen a number of different RNAi-targeted sequences from a target gene. Large-scale and systematic analyses of the specifi c features from the effective siRNAs reveal that siRNA might have sequence-specifi c features associated with its functionality. These molecular characteristics generally include low-to-medium G/C content (30-50%), high internal stability at the sense strand 5′-terminus, low internal stability at the sense strand 3′-terminus, absence of internal repeats or palindromes, and 6. Design the sense and antisense oligonucleotides for shRNA: shGene-S: N N N N N N N N N N N N N N N N N N t t c a a g a g a NNNNNNNNNNNNNNNNNNCTTTTTGGAAA-3′

and shGene-AS:

This reporter-based siRNA validation system includes two expression vectors for targeting reporter and triggering siRNA expressions ( see Fig. 1a, c ) . To fully normalize the transfection variation and accurately evaluate the effi cacy of the RNAi-targeted sequences, the targeting reporter vectors all contain two independent expression cassettes for transcription of the targeting reporter (enhanced green fl uorescence protein, EGFP, or fi refl y luciferase, Fluc) and reference protein (glutathione S-transferase, GST, or Renilla luciferase, Rluc) genes ( see Fig. 3a ). The easy and sensitive EGFP fl uorescence detection or Fluc activity assay combined with well-documented and easily analyzed reference protein, GST or Rluc, provides a simple and reliable readout for the system. To simply and effi ciently perform this reporter-based siRNA validation system, this method has only focused on the targeting reporter expression vectors, pEGFP-3′UTR and pFluc-3′UTR ( see Fig. 3b ), and the triggering siRNA and shRNA expression vectors, pDual and pHsH1 ( see Fig. 3c ). To enhance the convenience of constructing this reporter-based siRNA validation system and facilitate the screening of recombinant clones, all the vectors are further improved by inserting a stuffer of phosphoglycerate kinase (PGK) gene between the unique cloning sites, Bgl II and Hin dIII, which makes the preparation of the DNA vectors simple and easy by only removing the stuffer of PGK DNA fragment with Bgl II and Hin dIII double digestion ( see Note 1 ). To fully utilize the short synthetic DNA fragment for producing the triggering siRNA, we have constructed a particular siRNA expression vector, pDual ( see Fig. 3c ), which contains two convergent RNA Pol III promoters, mouse U6 and human H1, to drive the expression of both sense and antisense strands of siRNA using the short synthetic DNA fragment as template, respectively. In addition, to simply and effi ciently clone the short synthetic DNA fragment containing the RNAi-targeted sequence of interest into this vector, the pDual vector also contains the same Hin dIII and Bgl II restriction enzyme sites located between mouse U6 and human H1 promoters in which the sense and antisense strands of siRNA are transcribed by U6 and H1 promoters, respectively. It has been shown that shRNA exhibits slightly better effect on the inhibition of gene expression as compared with that of the siRNA [ 21 , 42 ] . To make this system complete, we have particularly used a highly effective shRNA expression vector, pHsH1 ( see Fig. 3c ) [ 41 ] , which contains only the human H1 promoter to drive transcription of the consecutive sequence of the sense, a loop, and the antisense regions.

In addition, to objectively and accurately examine the efficiency of selected RNAi-targeted sequence-mediated inhibition of the targeting reporter gene expression in both the EGFP-and Fluc-based siRNA validation systems, it is important to have highly effective RNAi-targeted sequences directly against EGFP and Fluc expressions. For this purpose, we have particularly constructed four effective siRNA and shRNA expression vectors for silencing EGFP (pDual-siEGFP and pHsH1-shEGFP) and Fluc (pDual-siFluc and pHsH1-shFluc) expressions that could serve as references for positive controls [ 41 ] . These effective RNAi-targeted sequences directly target on the coding regions of EGFP and Fluc mRNA transcripts and exhibit strong inhibition effects with a silencing effi ciency of more than 90% ( see Table 1 ).

1. Digest 10 μg of pEGFP-3′UTR-PGK, pFluc-3′UTR-PGK, or pDual-PGK in a 1.5 mL Eppendorf tube in a reaction with 5 μL of 10× restriction enzyme buffer, 10 units of Bgl II and Hin dIII, and distilled H 2 O to total 50 μL in 37 °C water bath for 2 h ( see Note 1 ).

2. Analyze 1 μL of digested DNA mixtures on a 0.8% (wt/vol) agarose gel with an appropriate molecular weight marker.

3. Inactivate the restriction enzymes by incubation on a 70 °C heat block for 10 min.

4. Isolate the digested vector by using electrophoresis on a 0.8% (wt/vol) agarose gel.

5. Recover the DNA fragment from the agarose gel by using the gel extraction kit, and elute the DNA fragment with 50 μL of TE (pH 8.0). Table 1 Sequences of synthetic DNA oligonucleotides used in this method for constructing siRNA validation system Name Synthetic DNA oligonucleotides

To conduct this reporter-based siRNA validation system, two complementary sense and antisense oligonucleotides were annealed to from a short synthetic DNA fragment with two unique enzyme-compatible ends Hind III and Bgl II, which could be used to insert into the Hind III/ Bgl II-digested targeting reporter and triggering siRNA expression vectors simultaneously 1. Mix 5 μL of the complementary oligonucleotides (100 μM) in a 1.5 mL Eppendorf tube in a reaction with 2 μL of 10× annealing buffer (T4 DNA ligase ligation buffer) and distilled H 2 O to total 20 μL ( see Note 5 ).

2. Place the Eppendorf tube in a 95 °C heat block for 10 min.

3. Remove the Eppendorf tube from the heat block and allow to cool to room temperature on the bench.

4. Spin down briefl y the Eppendorf tube to recover the reaction solution and store on ice at 4 °C until ready to use ( see Note 6 ). This sequencing can be carried out by an institutional core sequencing facility or a professional sequencing service.

Much evidence has already shown that not all of the RNAi-targeted sequences selected from a target gene display the same potencies on inducing gene silencing. Only a small number of siRNAs are capable of inducing highly effi cient target gene silencing in a sequence-specifi c manner. The silencing effi cacy of siRNAs is dependent on the specifi city of the target sites within the gene and can only be determined experimentally based on the inhibition of the target gene expression. Several widely used approaches can be utilized to analyze the effi ciency of gene silencing induced by DNA vector-based siRNA or shRNA expression, including (1) Northern blot, (2) quantitative reverse transcription (RT)-PCR or real-time

RT-PCR, (3) Western blot, (4) immuno-staining, and (5) functional activity assay. In general, the effect of gene silencing can be detected 24-48 h after transfection, dependent on the abundance and the stability of the proteins encoded by the target genes ( see Fig. 4 ).

1. Subculture and plate 1 × 10 5 cells per well in 2 mL growth medium onto a 6-well culture plate at 24 h before transfection. After 48-h incubation, the EGFP-expressed cells are fi rst examined under an inverted fl uorescence microscopy and then harvested and lysed for protein-level analysis by using Western blot. In addition, the luciferaseexpressed cells are harvested and lysed for protein-level analysis by using functional reporter assay (luciferase activity) directly against the novel or the putative genes by using this system. Once the highly effective RNAi-targeted sequences are identifi ed, one could easily establish the specifi c gene knockdown in the in vitro cell lines or in vivo animal models that provide a loss-of-function mutation of the novel or the putative genes. Subsequently, many different molecular, cellular, biochemical, and other analyses could be performed to examine the inhibition effects on the in vitro or in vivo models. siRNAs could be used clinically to inhibit gene expression as a therapeutic agent in many diseases characterized by elevated gene function. Inhibition of virus-specifi c genes by siRNAs has proven to be a potential therapeutic strategy against virus-induced diseases. A number of extremely virulent viruses including Ebola, Lassa, severe acute respiratory syndrome (SARS), avian infl uenza (H5N1), West Nile, and smallpox viruses are highly infectious and cause extraordinarily deadly diseases [ 43 , 44 ] . Furthermore, there are currently no vaccines or effective therapies available, and in particular these viruses require special containment for safe research. To develop an extremely potent RNAi-based therapeutics for these virulent viruses with safety, this reporter-based siRNA validation system could provide a simple and powerful approach for screening and identifi cation of highly effective siR-NAs directly against viral gene expression without the need of direct virus culture.

1. The main advantage of cloning procedures presented in this method is that preparation of the inserting vectors is simple and effi cient by only double digestion with restriction enzymes Bgl II and Hin dIII to remove the stuffer of PGK gene from pEGFP-3′UTR-PGK, pFluc-3′UTR-PGK, pDual-PGK, and pHsH1-PGK vectors. This will largely increase the cloning effi ciency to more than 75%.

2. The siRNA validation systems described in this method are cost effective and convenient in that any annealed oligonucleotide duplexes can be simultaneously cloned into both targeting reporter and triggering siRNA expression vectors.

3. The length of duplex region for a siRNA (shRNA) is relatively fl exible from 19 to 29 nt. Although increasing the length of duplex region for a relatively ineffective 19-nt siRNA (shRNA) can increase its effectiveness, increasing the length of an effective 19-nt siRNA (shRNA) may not further improve the inhibition effect.

4. The oligonucleotides used for constructing the systems can be purchased from any local commercial suppliers without any further modifi cation or treatment.

5. The annealing of two complementary oligonucleotides can be effi ciently carried out in 1× T4 DNA ligase buffer, which can be obtained from any T4 DNA ligase commercial suppliers. 6 . The annealed oligonucleotide duplexes should not be phosphorylated before the ligation step, because it might result in multiple copies of insertion.

7. Using this protocol for cloning the targeting reporter and triggering siRNA or shRNA expression cassettes is effi cient and cost effective in that only four colonies are selected and screened for the positive clones containing the RNAi-targeted sequence, siRNA or shRNA expression sequence.

Protein lysis buffer: 50 mM NaCl, 50 mM Tris-HCl

Protease inhibitors

Micro bicinchoninic acid (Micro BCA) assay

Bovine serum albumin (BSA

Dual-luciferase Reporter Assay System (Promega) stored in aliquots at −80 °C freezer

Enhanced chemiluminescence (ECL) Western blotting detection reagents

UV image system (UV illuminator

Microplate reader (Dynatech MR5000; Dynatech Laboratories

MiniLumat LB 9506

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus (Mighty Small II 8 × 7 cm

Potent and specifi c genetic interference by double-stranded RNA in Caenorhabditis elegans

Mechanisms of gene silencing by double-stranded RNA

Role for a bidentate ribonuclease in the initiation step of RNA interference

RNase III enzymes and the initiation of gene silencing

Single-stranded antisense siRNAs guide target RNA cleavage in RNAi

Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways

Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing

A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi

RNAimediated pathways in the nucleus

Functional genomics: RNA sets the standard

A large-scale RNAi screen in human cells identifi es new components of the p53 pathway

A resource for large-scale RNA-interferencebased screens in mammals

RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age

RNAi: gene-silencing in therapeutic intervention

Unlocking the potential of the human genome with RNA interference

Approaches for the sequence-specifi c knockdown of mRNA

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

siRNAs: applications in functional genomics and potential as therapeutics

Synthetic shRNA as potent RNAi triggers

A system for stable expression of short interfering RNAs in mammalian cells

A DNA vectorbased RNAi technology to suppress gene expression in mammalian cells

Short hairpin RNAs (shRNAs) induce sequence-specifi c silencing in mammalian cells

Effective expression of small interfering RNA in human cells

Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells

U6 promoterdriven siRNAs with four uridine 3′ overhangs effi ciently suppress targeted gene expression in mammalian cells

An approach to genomewide screens of expressed small interfering RNAs in mammalian cells

Rational siRNA design for RNA interference

Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides

siRNA target site secondary structure predictions using local stable substructures

Functional siRNAs and miRNAs exhibit strand bias

Asymmetry in the assembly of the RNAi enzyme complex

Improving the effi ciency of RNA interference in mammals

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

Highthroughput selection of effective RNAi probes for gene silencing

Validating siRNA using a reporter made from synthetic DNA oligonucleotides

A rapid and sensitive assay for quantifi cation of siRNA effi ciency and specifi city

A novel siRNA validation system for functional screening and identifi cation of effective RNAi probes in mammalian cells

Simple and efficient DNA vector-based RNAi systems in mammalian cells

Emerging viral infections in a rapidly changing world

The challenge of emerging and re-emerging infectious diseases

This work was supported by grants from the National Science Council of Taiwan, ROC (to Wen-Tsan Chang).

2. Co-transfect 0.5 μg of targeting reporter and 1.5 μg of triggering siRNA expression vectors by using Lipofectamine 2000 following the manufacturer's protocol. 3 . Incubate the transfected cells at 37 °C in a CO 2 incubator for 48 h.1. Remove growth medium, and wash the transfected cells three times with PBS.2. Examine the expressed EGFP in cells by using inverted fl uorescence microscopy.3. Confi rm the expression levels of EGFP and GST in the total protein extracts by using Western blot analysis.1. Remove growth medium, and wash the transfected cells three times with PBS.2. Harvest the transfected cells from the plate by using cell scrapers or spatulas into a 50 mL culture tube.3. Prepare total cell lysates from the transfected cells by using protein lysis buffer containing protease inhibitors.4. Perform Western blot analysis with specifi c antibodies for EGFP and GST according to standard protocols.1. Remove growth medium, and wash the transfected cells three times with PBS.2. Harvest the transfected cells from the plate by using cell scrapers or spatulas into a 50 mL culture tube.3. Prepare total cell lysates from the transfected cells by using cell lysis buffer.4. Perform the dual-luciferase reporter assay system as described by the manufacturer.This system is composed of two expression vectors for targeting reporter and triggering siRNA expressions as well as two highly effective siRNAs, siEGFP and siFluc, which serve as references for positive controls [ 41 ] . As compared with the inhibition levels of these positive references, one could easily evaluate the effi cacy of selected and designed RNAi-targeted sequences of interest.There are many strategies to build the profi le of a gene and its function. One of the best and simple ways to elucidate gene function is to disrupt or inhibit the gene and characterize the phenotype of resulting mutant. To effi ciently apply RNAi technology for reverse functional genomics [ 12 -15 ] , in particular the novel or the putative genes with only available nucleotide sequences in databases but without cDNA clones in hand, one could simply identify the effective RNAi-targeted sequences 3