Inducing gene expression by targeting promoter sequences using small activating RNAs
2 Biotechnology Research Institute and Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
3 Laboratory of Molecular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
Promoter analysis and gene selection
saRNA design and target location
Methods for introducing saRNAs into cells
Cell preparation for transfection
saRNA concentrations and related kinetics
Control treatments and experimental replication
1.Control saRNAs. Control saRNAs can be composed of unrelated or scrambled sequence in relationship to a functional saRNA. It should be noted that RNAa does not require perfect complementarity between the saRNA and its target sequence to function . As such, controls should noticeably deviate from functional saRNA sequence. Additionally, control duplexes should be checked for potential homology with sequence in the genome of the same organism in order to limit putative function. BLAST searches can be performed by selecting the RefSeq RNA or the reference genome database. A sequence that returns hits with significant homology (≥ 80%), particularly if it matches known transcripts or sequence neighboring the targeted promoter, should be rejected as controls. Ideally, control duplexes should possess similar GC content and terminal thermodynamic asymmetry as the functional saRNA. Testing RNAa activity against multiple control duplexes also enriches validity of saRNA experiments.
2.Transfection controls. Transfections in absence of duplex RNA can be a very useful control for identifying any effects on cell viability or gene expression related to the use of transfection reagents.
3.Assessment of transcriptional gene activation. The technique of RNAa described in this report occurs at the level of transcription. To distinguish from post-transcriptional mechanisms that can lead to elevated mRNA levels, there are several experimental options that may be performed to confirm transcriptional activation. For instance, nuclear run-on is considered the gold standard for accessing transcriptional upregulation at a specific target gene. Additionally, enrichment of RNAPII can be quantified at the target gene promoter by ChIP analysis as a marker for transcriptional activation. Measuring levels of pre-mRNA by RT-PCR can also be used as a method to assess changes in gene transcription .
4.Multiple saRNAs targeting the same promoter. Identifying multiple saRNAs that activate gene expression at different positions (e.g. dsEcad-215 and -302) improves the likelihood that gene induction is dependent on the targeted promoter (Fig. 1A-C). Each activating duplex likely possesses a different ‘seed’ sequence and target non-overlapping regions. As such, the probability for each duplex functioning by suppression of non-specific upstream regulators becomes increasing lower. Additionally, if changes in molecular and/or cellular downstream phenotypes are conserved between multiple saRNAs, it correlates with gene activation being on-target.
5.Verifying target specificity by vector-mediated overexpression. Ectopic expression of genes using vector-based systems remains the standard method for gain-of-function study. The specificity of downstream gene modulation and functional consequences following RNAa can be verified by vector-mediated overexpression of the same gene. Recapitulation of RNAa by an ectopic system can validate the phenotypic changes facilitated by saRNAs [32,45].
6.Evaluating candidate off-target gene expression. Even carefully selected saRNAs or siRNAs cannot avoid partial sequence homology with other coding and non-coding sequences. Partial homology with off-target coding sequence may trigger miRNA-like mechanisms of post-transcriptional gene silencing. As such, expression of putative off-target genes should be screened to remove the potential for non-specific mechanisms of gene activation.
- Synthetic short duplex RNA (desalted or HPLC purified)
- Diethyl pyrocarbonate (DEPC, Sigma, cat. no. 40718). CAUTION: Toxic. Use personal protective equipment and avoid breathing vapors, mist or gas
- RPMI-1640 medium (Invitrogen, cat. no. 11875-093)
- Opti-MEM® I Reduced Serum Medium (Invitrogen, cat. no. 31985-062)
- 1×Trypsin-EDTA (0.05% Trypsin with EDTA 4Na) 1× (Invitrogen, cat. no. 25300-054)
- Lipofectamine™ RNAiMax™ transfection reagent (Invitrogen, cat. no. 13778-150)
- Lipofectamine 2000™ transfection reagent (Invitrogen, cat. no. 11668-019)
- Silencer® Cy™3 labeled Negative Control No. 1 siRNA (Applied Biosystems, cat. no. AM4621)
- GelRed (Biotium, cat. no. 41003)
- Power SYBR Green PCR Master Mix (2X) (Applied Biosystems, cat. no. 4367659)
- TaqMan® Fast Universal PCR Master Mix (Applied Biosystems, cat. no. 4352042)
- Reagents for standard cell lysis and protein quantification
- RIPA buffer with EDTA (Boston BioProducts, cat. no. BP-115D)
- Protease inhibitor cocktail (Sigma, cat. no. P8340)
- BCA protein assay reagent (bicinchoninic acid; Thermo Scientific, cat. no. 23227)
- Reagents for standard SDS-PAGE and western blotting assay:
- 30% Acrylamide/Bis solution, 29:1 (Bio-Rad, cat. no. 161-0156)
- APS (ammonium persulfate; Bio-Rad, cat. no. 161-0700)
- TEMED (N,N,N′,N′-tetramethylethylenediamine; Sigma, cat. no. T22500)
- Trizma base (Sigma, cat. no. T6066)
- Glycine (USB, cat. no. 16407)
- Methanol (Fisher Scientific, cat. no. A412)
- SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, cat. no. 34087)
- Sterile 6-well cell culture plates (Nunc, cat. no. 140685)
- Sterile 15 ml conical tubes (Santa Cruz Biotechnology, cat. no. sc-200250)
- 1.5-ml microcentrifuge tubes (USA Scientific, cat. no. 1615-5510)
- Standard cell culture equipment including laminar flow hood, humidified tissue culture incubators at 37°C and 5% CO2, water bath, refrigerator and -20°C freezer.
- Refrigerated centrifuge with swing-bucket rotor accommodating 15-ml conical tubes.
- Inverted microscope with combined ocular-objective magnification of 40×, 100× and 250× and camera
- 7500 Fast Real-Timer PCR System (Applied Biosystems, cat. no. 4351107)
- Standard molecular biology plasticware and glassware
- UV transilluminator
- Agarose gel electrophoresis apparatus
- Single-channel pipettes (2 µl, 10 µl, 100 µl and 1000 µl) with arousal resistant plastic pipette tips
- NanoDrop 2000 (Thermo Scientific)
- Personal computer with Internet access and web browser
- Sequence analysis and manipulation program, or text editor
- Equipment for standard cell lysis, SDS-PAGE and western blotting assay including refrigerated centrifuge such as centrifuge Legend RT (Thermo Scientific, cat. no. 75-004-377), spectrophotometer such as Multiskan MCC/340 (Thermo Scientific, cat. no. 14-386-26), vertical electrophoresis systems such as Mini Protean Tetra Cell (Bio-Rad, cat. no. 166-0828EDU), shaker such as Nutator Mixer (BD, cat. no. 421105), nitrocellulose membranes such as NitroBind (GE Water & Process Technologies, EP4HY00010), film cassette such as autoradiography cassettes (Fisher Scientific, cat. no. FB-CS-810), and autoradiography film such as HyBlot CL (Denville scientific, cat. no. E3018).
Promoter identification and sequence retrieval (1 h)
1.Using the Ensembl Genome Browser (EGB)
1.1.Go to EGB at http://www.ensembl.org
1.2.Choose the desired genome in the search box and type the name or symbol of the intended target gene.
1.3.On the result page, choose the correct gene by clicking “Gene” under the category “By Feature type”.
1.4.On the “Result in Detail” page, click the title of the correct gene.
1.5.Click “Sequence” on the left column, which will lead to the marked-up sequence page. On the lower half of the page, a 600-bp (by default) 5’ flanking sequence, exons, introns, 3’ UTR, and 3’ flanking sequence are displayed with exons in red text.
1.6.To display 1 kb of the 5’ flanking sequence of the gene, click “Configure this page” on the lower left column and a new window will pop up. In the text box for “5’ Flanking sequence (upstream)”, type “1000” and click the check (√) sign on the upper right hand corner of the window to save the change and close the window.
1.7.The marked-up sequence page will reload to reflect the change by displaying 1 kb 5’ flanking sequence at the beginning followed by the genomic sequence for the rest of the gene.
1.8.Copy the first 1 kb sequence which is the putative promoter sequence of the gene and paste it into a text editing program.
2.Using the UCSC Genome Browser (UGB)
2.1.Go to UGB at http://genome.ucsc.edu/cgi-bin/hgGateway
2.2.Choose the desired genome by first selecting clade and then genome in the dropdown menu of the search box.
2.3.In the box “position or search term”, type the name or symbol of the intended target gene and click “submit”.
2.4.In the result page, under RefSeq genes or UCSC genes, click the correct gene. The next page will display the genomic structure of the gene along with many different annotation tracks.
2.5.Click “View” then “ DNA” on the top menu bar, and type “1000” in the box for “Add [ ] extra bases upstream (5’)” and then click “get DNA”.
2.6.Copy the first 1 kb sequence as the promoter sequence of the gene and paste into a text editing program.
Promoter sequence analysis (1 h)
3.Identify CpG islands in the retrieved promoter sequence using UGB CpG island annotation or other online programs such as MethPrimer at http://www.urogene.org/cgi-bin/methprime, EMBOSS CpGPlot/CpGReport at http://www.ebi.ac.uk/Tools/emboss/cpgplot.
4.Identify repetitive sequences and sequence variants by examining the sequence in UGB’s “Variation and Repeats tracks” or using the RepeatMasker Web Server at http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker.
saRNA target selection (1-2 h)
5.Manually saRNA target selection
5.1.Paste the 1-kb promoter sequence into a sequence analysis program or any text editor.
5.2.Starting from -100 bp, visually examine the sequence toward its 5’ end and select targets based on the following rules (Table 1):
5.2.1.Use the sense DNA sequence of the promoter as the template for saRNA design.
5.2.2.Targets can be selected within a promoter region between at -100 and -1000 bp upstream the TSS.
5.2.3.Targets should be 19-nt in size.
5.2.4.Targets should have a GC content of 40-60%, GC-rich regions or CpG islands should be avoided.
5.2.5.The corresponding saRNA duplexes should have lower thermodynamic stability at the 3’ end than the 5’ end (Fig. 2).
5.2.6.The 18th and 19th positions counted from the 5’ end of targets should be “A/T”s, preferably “A”s.
5.2.7.Avoid sequences that have 5 or more consecutive nucleotides.
5.2.8.Avoid simple repeat sequences such as di- or tri-nucleotide repeats.
5.2.9.The 20-23th nucleotides (flanking the 3’ end of a target) should preferably be “A”s or “T”s.
5.3.Repeat Step 5 to choose 4-5 targets.
6.Select saRNA targets using an Excel program which implements the above rules (File S1).
6.1.Download the Excel program (File S1) and save it on a local computer.
6.2.Open the Excel file and choose “Allow Macro” when prompted.
6.3.Paste the 1-kb promoter sequence into the DNA sequence input box.
6.4.Optional: Specify the location of the TSS if it is known.
6.5.Click the button “Click here to find the best saRNAs”.
6.6.Choose 4–5 saRNA targets based on overall quality score.
7.For each candidate target, run a BLAST search by going to http://blast.ncbi.nlm.nih.gov/Blast.cgi.
8.Use the 19-nt target sequence as query sequence, “Reference genome sequences” as “Search Set”, and “Optimize for Highly similar sequences (megablast)” as “Search Program”.
9. Use the 19-nt target sequence along with 1 nt 5’ or 3’ flanking sequence as query sequence to run a BLAT search (http://genome.ucsc.edu/cgi-bin/hgBlat) to identify SNPs within the target and reject the target if it contains SNPs.
10.Once a 19-nt target is selected, create a sense RNA sequence based on the target sequence by converting all ‘T’s to ‘U’s in the target sequence and an antisense RNA sequence by reverse complementing the sense RNA sequence.
11.Add either [dT][dT] or [U][U] overhangs to the 3’-end of both RNA strands.
12.Perform chemical synthesis of the RNA strands and anneal them to make a dsRNA duplex, or order the dsRNA duplex from a reliable vendor.
saRNA transfection of cells using a reverse transfection protocol (3–4 d)
13.Prepare one tube (Tube A, for diluting saRNAs) for each well to be transfected, and one master mixture tube (Tube B, for diluting transfection reagent) for all wells.
14.Remove RNAiMAX from the refrigerator and let it warm up to room temperature.
15.Before use, gently mix RNAiMAX solution by tapping on the tube.
16.Add 250 µl of Opti-MEM medium and 5 µl RNAiMAX to tube B, mix gently by tapping on the tube or pipetting up and down a few times. A master mixture can be prepared in Tube B for all treatments.
17.Add 250 µl Opti-MEM medium to each of Tube As.
18.Add 6.25 µl of 20 µM saRNA to each of Tube As and mix well. For mock transfection, omitting dsRNA.
19.Add 250 µl of the diluted RNAiMAX from tube B into each of Tube As. Mix well by pipetting or inverting the tubes several times.
20.Let the tube containing both diluted saRNA and diluted RNAiMAX set at room temperature for 10–20 min to allow transfection complex to form.
21.If a transfection reagent other than RNAiMAX is used, follow manufacturer’s manual.
22.Remove medium from exponentially growing cells in a 10-cm dish.
23.Wash cells with 1–2 ml PBS and remove the PBS.
24.Add 1 ml 0.05% trypsin into the dish and rock the dish to allow for even coverage of the surface by trypsin.
25.Return the dish to a CO2 incubator and wait for 5 min.
26.While waiting, add 10 ml fresh complete medium into a 15-ml conical tube.
27.Remove the dish from the incubator after 5 min (or shorter to prevent over-digestion). Pipet up and down the trypsin solution containing digested cells to disperse cells into single cell suspension.
28.Transfer cells to the 15-ml conical tube containing 10 ml complete medium.
29.Mix the cells with the complete medium by pipetting.
30.Take 100 µl of the mixed cell suspension and add it to a 1.5-ml microcentrifuge tube containing 900 µl PBS to dilute the cells, mix well for cell counting.
31.Centrifuge the remaining cells in the 15-ml tube at 800 × g and 4°C for 5 min.
32.While waiting, count the cells using a hemocytometer and calculate the total number of cells in the 15-ml tube.
33.When centrifugation is done, remove the supernatant from the tube and resuspend cells in the desired amount of medium without antibiotics that gives the desired cell concentration.
34.Mix the cells well and add 2 ml of the cells suspension to each well.
35.Gently swing the plate to distribute the cells evenly in the wells.
36.Return the plate to the incubator if the transfection mixture is still incubating.Adding transfection complex to the cells
37.Add the transfection mixture to each well drop-wisely and evenly to all areas of the well.
38.Check under a microscope to make sure that the cells are evenly distributed across the surface of every well; otherwise, swing the plate again to distribute the cells evenly.
39.Return the plate to the incubator.
40.Optional: Change the transfection medium with fresh complete medium 24 h later.
41.Observe and document cell morphology using an inverted microscope equipped with a camera starting from 48 h when RNAa effect begins to appear.
RNA isolation and reverse transcription (RT) reaction (1 d)
42.Isolate total cellular RNA using a method or kit of choice.
43.Treat the RNA with DNase I to remove potential DNA contamination.
44.Accurately measure RNA concentrations using Nanodrop 2000 or a UV spectrophotometer.
45.Use 100–1000 ng of the isolated RNA in RT reaction along with oligo(dT) or random hexamer primers, RNase inhibitor, dNTP, reverse transcriptase with compatible buffer in a 25–30 µl RT reaction to convert the RNA into cDNA.
46.Dilute the resulted cDNA (1:4) using nuclease-free water.
Analysis of gene activation at the mRNA level by semi-quantitative and quantitative RT-PCR (1 d)
47.mRNA expression analysis by semi-quantitative RT-PCR.
47.1.Using the diluted cDNA as template to amplify mRNA of the target gene using gene specific RT-PCR primers by semi quantitative RT-PCR in a PCR thermocycler. A housekeeping gene should also be amplified as RNA loading controls.
47.2.Use 1–2% agarose gel prestained with GelRedtm Stain to separate the PCR products.
47.3.Visualize and document the gel image using a UV transilluminator at 254 nm.
48.mRNA expression analysis by quantitative RT-PCR (qRT-PCR). To quantify mRNA expression levels of the target gene and a housekeeping gene, use standard SYBR® green or TaqMan® based detection using a real-time thermal cycler.
Analysis of gene activation at the protein level by standard western blotting assay (1–2 d)
49.Collect cells by scraping, wash cell pellet twice with PBS and lyse cells with RIPA buffer with protease inhibitor cocktail for 30 min on ice. Centrifuge lysates for 15 min at 14,000 rpm 4°C and collect supernatants.
50.Optional: If activation of a target gene is expected to cause a decrease in cell proliferation than control treatments such as the activation of p21 gene , combine multiple wells of saRNA treated cells to obtain enough cell lysate.
51.Determine protein concentration by BCA protein assay.
52.Load equal amounts of total protein in lysates (30–50 μg) onto a SDS-PAGE gel and transfer the separated proteins to a nitrocellulose membrane.
53.Block the membrane in 5% nonfat milk and incubate with primary antibodies at 4°C overnight. After incubation with horseradish peroxidase-conjugated secondary antibodies, detect signals by chemiluminescent reagents.
|Distance from TSS||-100 – -1000|
|Target size||19 nt|
|Target GC content||40–60%|
|Consecutive nucleotides||<= 5|
|Simple repeats (di or tri-nucleotide)||avoid|
|CpGs in target||<= 1|
|1st and 2nd nucleotides||preferably G or C|
|18th nucleotide||A or T|
|20–23th nucleotides||preferably A or T|
|14–53||Low or no RNAa activity||Transfection efficiency is too low||
|Transfection duration is too short.||
|Use of a suboptimal number of cells.||
|Inappropriate target design||
|Gene silencing by epigenetic mechanisms especially DNA methylation||
|13–53||RNAa is not reproducible||dsRNA degradation||
|Different passages of primary cells or cell lines are used||
|49–53||RNAa cannot be confirmed by protein expression analysis||Use un-validated primary antibodies for target protein detection.||
|Primary antibody does not recognize the specific isoform induced by promoter-targeted saRNAs.||
Author contributions statements
- Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. doi: 10.1038/35888. [View Article] [PubMed] [Google Scholar]
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498. doi: 10.1038/35078107. [View Article] [PubMed] [Google Scholar]
- Song E, Lee S, Wang J, Ince N, Ouyang N, et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9: 347-351. doi: 10.1038/nm828. [View Article] [PubMed] [Google Scholar]
- Davis ME, Zuckerman JE, Choi CHJ, Seligson D, Tolcher A, et al. (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464: 1067-1070. doi: 10.1038/nature08956. [View Article] [PubMed] [Google Scholar]
- Morris KV, Chan SW, Jacobsen SE, Looney DJ (2004) Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305: 1289-1292. doi: 10.1126/science.1101372. [View Article] [PubMed] [Google Scholar]
- Janowski BA, Huffman KE, Schwartz JC, Ram R, Hardy D, et al. (2005) Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nat Chem Biol 1: 216-222. doi: 10.1038/nchembio725. [View Article] [PubMed] [Google Scholar]
- Yue X, Schwartz JC, Chu Y, Younger ST, Gagnon KT, et al. (2010) Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini. Nat Chem Biol 6: 621-629. doi: 10.1038/nchembio.400. [View Article] [PubMed] [Google Scholar]
- Li LC, Okino ST, Zhao H, Pookot D, Place RF, et al. (2006) Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A 103: 17337-17342. doi: 10.1073/pnas.0607015103. [View Article] [PubMed] [Google Scholar]
- Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, et al. (2007) Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 3: 166-173. doi: 10.1038/nchembio860. [View Article] [PubMed] [Google Scholar]
- Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R (2008) MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A 105: 1608-1613. doi: 10.1073/pnas.0707594105. [View Article] [PubMed] [Google Scholar]
- Matsui M, Sakurai F, Elbashir S, Foster DJ, Manoharan M, et al. (2010) Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter. Chem Biol 17: 1344-1355. doi: 10.1016/j.chembiol.2010.10.009. [View Article] [PubMed] [Google Scholar]
- Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6: 211-221. doi: 10.1016/j.celrep.2013.12.013. [View Article] [PubMed] [Google Scholar]
- Chu Y, Yue X, Younger ST, Janowski BA, Corey DR (2010) Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter. Nucleic Acids Res 38: 7736-7748. doi: 10.1093/nar/gkq648. [View Article] [PubMed] [Google Scholar]
- Turunen MP, Lehtola T, Heinonen SE, Assefa GS, Korpisalo P, et al. (2009) Efficient regulation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism: a novel example of epigenetherapy. Circ Res 105: 604-609. doi: 10.1161/CIRCRESAHA.109.200774. [View Article] [PubMed] [Google Scholar]
- Gagnon KT, Corey DR (2012) Argonaute and the nuclear RNAs: new pathways for RNA-mediated control of gene expression. Nucleic Acid Ther 22: 3-16. doi: 10.1089/nat.2011.0330. [View Article] [PubMed] [Google Scholar]
- Portnoy V, Huang V, Place RF, Li LC (2011) Small RNA and transcriptional upregulation. Wiley Interdiscip Rev RNA 2: 748-760. doi: 10.1002/wrna.90. [View Article] [PubMed] [Google Scholar]
- Place RF, Noonan EJ, Földes-Papp Z, Li LC (2010) Defining features and exploring chemical modifications to manipulate RNAa activity. Curr Pharm Biotechnol 11: 518-526. [PubMed] [Google Scholar]
- Huang V, Qin Y, Wang J, Wang X, Place RF, et al. (2010) RNAa is conserved in mammalian cells. PLoS One 5: doi: 10.1371/journal.pone.0008848. [View Article] [PubMed] [Google Scholar]
- Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD (1988) Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A 85: 836-840. [PubMed] [Google Scholar]
- Clark AJ, Archibald AL, McClenaghan M, Simons JP, Wallace R, et al. (1993) Enhancing the efficiency of transgene expression. Philos Trans R Soc Lond B Biol Sci 339: 225-232. doi: 10.1098/rstb.1993.0020. [View Article] [PubMed] [Google Scholar]
- Stemmler MP, Hecht A, Kemler R (2005) E-cadherin intron 2 contains cis-regulatory elements essential for gene expression. Development (Cambridge, England 132: 965-976. doi: 10.1242/dev.01662. [View Article] [PubMed] [Google Scholar]
- Breitbart RE, Nguyen HT, Medford RM, Destree AT, Mahdavi V, et al. (1985) Intricate combinatorial patterns of exon splicing generate multiple regulated troponin T isoforms from a single gene. Cell 41: 67-82. [PubMed] [Google Scholar]
- Leff SE, Rosenfeld MG, Evans RM (1986) Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem 55: 1091-1117. doi: 10.1146/annurev.bi.55.070186.005303. [View Article] [PubMed] [Google Scholar]
- Hedman M, Hartikainen J, Yla-Herttuala S (2011) Progress and prospects: hurdles to cardiovascular gene therapy clinical trials. Gene Ther 18: 743-749. doi: 10.1038/gt.2011.43. [View Article] [PubMed] [Google Scholar]
- Boguski MS, Lowe TM, Tolstoshev CM (1993) dbEST--database for "expressed sequence tags. Nat Genet 4: 332-333. [PubMed] [Google Scholar]
- Wakaguri H, Yamashita R, Suzuki Y, Sugano S, Nakai K (2008) DBTSS: database of transcription start sites, progress report 2008. Nucleic Acids Res 36: 97-101. doi: 10.1093/nar/gkm901. [View Article] [PubMed] [Google Scholar]
- Larsen F, Gundersen G, Lopez R, Prydz H (1992) CpG islands as gene markers in the human genome. Genomics 13: 1095-1107. [PubMed] [Google Scholar]
- Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18: 1427-1431. [PubMed] [Google Scholar]
- Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, et al. (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22: 326-330. doi: 10.1038/nbt936. [View Article] [PubMed] [Google Scholar]
- Ting AH, Schuebel KE, Herman JG, Baylin SB (2005) Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 37: 906-910. doi: 10.1038/ng1611. [View Article] [PubMed] [Google Scholar]
- Xia T, SantaLucia Jr, J. , Burkard ME, Kierzek R, Schroeder SJ, et al. (1998) Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37: 14719-14735. doi: 10.1021/bi9809425. [View Article] [PubMed] [Google Scholar]
- Wang J, Place RF, Huang V, Wang X, Noonan EJ (2010) Prognostic Value and Function of KLF4 in Prostate Cancer: RNAa and Vector-Mediated Overexpression Identify KLF4 as an Inhibitor of Tumor Cell Growth and Migration. Cancer Res 70: 10182-10191. doi: 10.1158/0008-5472.CAN-10-2414. [View Article] [PubMed] [Google Scholar]
- Chen Z, Place RF, Jia Z, Pookot D, Dahiya R, et al. (2008) Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells. Mol Cancer Ther 7: 698-703. doi: 10.1158/1535-7163.MCT-07-2312. [View Article] [PubMed] [Google Scholar]
- Wang J, Huang V, Ye L, Bárcena A, Lin G, et al. (2014) Identification of Small Activating RNAs that Enhance Endogenous OCT4 Expression in Human Mesenchymal Stem Cells. Stem Cells Dev 24: 345-353. doi: 10.1089/scd.2014.0290. [View Article] [PubMed] [Google Scholar]
- Yamashita R, Sathira NP, Kanai A, Tanimoto K, Arauchi T, et al. (2011) Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res 21: 775-789. doi: 10.1101/gr.110254.110. [View Article] [PubMed] [Google Scholar]
- Saxonov S, Berg P, Brutlag DL (2006) A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A 103: 1412-1417. doi: 10.1073/pnas.0510310103. [View Article] [PubMed] [Google Scholar]
- Anderson JD, Widom J (2001) Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites. Mol Cell Biol 21: 3830-3839. doi: 10.1128/MCB.21.11.3830-3839.2001. [View Article] [PubMed] [Google Scholar]
- Field Y, Kaplan N, Fondufe-Mittendorf Y, Moore IK, Sharon E, et al. (2008) Distinct modes of regulation by chromatin encoded through nucleosome positioning signals. PLoS Comput Biol 4: doi: 10.1371/journal.pcbi.1000216. [View Article] [PubMed] [Google Scholar]
- Chen R, Wang T, Rao K, Yang J, Zhang S, et al. (2011) Up-regulation of VEGF by small activator RNA in human corpus cavernosum smooth muscle cells. J Sex Med 8: 2773-2780. doi: 10.1111/j.1743-6109.2011.02412.x. [View Article] [PubMed] [Google Scholar]
- Nakayama A, Sato M, Shinohara M, Matsubara S, Yokomine T, et al. (2007) Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system. Cloning Stem Cells 9: 523-534. doi: 10.1089/clo.2007.0021. [View Article] [PubMed] [Google Scholar]
- Kang MR, Yang G, Place RF, Charisse K, Epstein-Barash H, et al. (2012) Intravesical delivery of small activating RNA formulated into lipid nanoparticles inhibits orthotopic bladder tumor growth. Cancer Res 72: 5069-5079. doi: 10.1158/0008-5472.CAN-12-1871. [View Article] [PubMed] [Google Scholar]
- Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, et al. (2010) A status report on RNAi therapeutics. Silence 1: 14. doi: 10.1186/1758-907X-1-14. [View Article] [PubMed] [Google Scholar]
- Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17: 3011-3016. doi: 10.1101/gad.1158803. [View Article] [PubMed] [Google Scholar]
- Schwartz JC, Corey DR (2011) Practical considerations for analyzing antigene RNAs (agRNAs): RNA immunoprecipitation of argonaute protein. Methods Mol Biol 764: 301-315. doi: 10.1007/978-1-61779-188-8_20. [View Article] [PubMed] [Google Scholar]
- Wang X, Wang J, Huang V, Place RF, Li LC (2012) Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation. Biochem J 443: 821-828. doi: 10.1042/BJ20111491. [View Article] [PubMed] [Google Scholar]
- Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, et al. (2001) Beta-actin--an unsuitable internal control for RT-PCR. Mol Cell Probes 15: 307-311. doi: 10.1006/mcpr.2001.0376. [View Article] [PubMed] [Google Scholar]