Molecular and Cellular Probes 28 (2014) 181e185

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Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method José A. Reyes-Darias, Alfredo Berzal-Herranz* Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC (IPBLN-CSIC), PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 December 2013 Accepted 21 February 2014 Available online 4 March 2014

Transfection of mammalian cells or in vivo administration of nucleic acids can induce inflammatory cytokines and/or interferon response, which could significantly influence the ex vivo or in vivo applications of gene-targeting strategies based on nucleic acids. Further induction of the interferon and inflammatory related stress responses may result in off-target effects and toxicity. This work describes an original one-step multiplex reverse transcription polymerase chain reaction procedure, which allows testing the induction of interferon and proinflamatory related responses by nucleic acids in the cell system of choice. The developed procedure has been tested on mammalian cells transfected with ssRNA, dsRNA, enzymatically synthesized siRNA and synthetic oligodesoxyribonucleotides containing unmethylated cytosine-guanosine motifs. This procedure is a rapid and convenient screening assay that could be used routinely in both the clinical and the research laboratory to validate the stimulation of the immune system on mammalian cells by nucleic acids. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Immune response detection Interferon Multiplex RT-PCR CpG DNA oligonucleotide siRNAs Toll-like receptors

1. Introduction Small interfering RNAs (siRNAs), antisense RNAs and DNA oligonucleotides (ODNs) that mediates specific gene silencing are widely used to study gene function and are also being developed for therapeutic applications [1]. However, many nucleic acids, including double-stranded RNA (dsRNA) [2], single-stranded RNA (ssRNA) [3e5], bacterial DNA and synthetic ODN containing unmethylated cytosine-guanosine motifs (CpG ODN) [6,7], can stimulate innate immune response in mammals. This could significantly influence the ex vivo and in vivo applications of these gene-targeting strategies owing to off-target effects and toxicities associated with immune stimulation. Innate immune responses can be triggered through a variety of so-called pattern recognition receptors, including Toll-like receptors (TLRs) that are activated by PAMPs (pathogen-associated molecular patterns) such as lipoproteins/lipopeptides, flagellin, lipopolysaccharides, and nucleic acids of bacteria and viruses [8,9]. The human TLR3 is the receptor for viral and synthetic dsRNAs [2], TLR7 and TLR8 are the receptors for viral ssRNAs and nucleosides [3,4,10] and TLR9 recognizes nomethylated CpG motifs of synthetic oligonucleotides, bacterial and

* Corresponding author. Tel.: þ34 958 18 16 48; fax: þ34 958 18 16 32. E-mail address: [email protected] (A. Berzal-Herranz). http://dx.doi.org/10.1016/j.mcp.2014.02.005 0890-8508/Ó 2014 Elsevier Ltd. All rights reserved.

viral DNA [11]. It has been shown that human TLRs are differentially expressed in a variety of tissues [12e15]. Agonists of TLRs 3, 7, 8, and 9 have been found to be potent inducers of alpha interferon (aINF) and the interferon-induced antiviral biomarker 20 ,50 -oligoadenylate synthetase (OAS) [16]. Activation of TLR9 induces IL-8 secretion in human cells [17]. ISG56 is a member of a family of proteins whose expression is strongly induced from very low basal levels by IFN [18]. The sensitivity of different cell lines to nucleic acids activation interferon-induced stress genes and pathways can vary significantly. Cell type, as well as growth conditions and passage number, can affect the susceptibility, level, and extend of activation of the interferon response. Therefore, a method that analyzes simultaneously a large number of genes or proteins is more likely to ensure the induction of an immune response. Currently, there are several methods available for detecting interferon response as an enzymelinked immunosorbent assay (ELISA) [19], western blotting, or a commercial monospecific reverse transcription polymerase chain reaction (RT-PCR) method (Interferon Response Detection Kit from System Biosciences). However these methods are laborious and time consuming. A multiplex RT-PCR technique that allows determining simultaneously the presence or absence of several specific RNA templates from RNA mixtures in a single reaction tube, is shown as the most efficient methodology for determining the stimulation of the immune system by nucleic acids. We have developed a multiplex RT-PCR method that allows the

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simultaneous detection of IFNa, ISG56, OAS2 genes involved in the interferon response and the pro-inflammatory cytokine IL-8 gene, a marker of a non-interferon mediated cellular response. In addition the glyceraldehyde-3-phosphate-dehidrogenase (GAPDH) is also detected as internal reference gene. 2. Materials and methods 2.1. Cell culture and transfection The human embryonic kidney (HEK) 293T cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM; PAA Laboratories GmBh, Cölbe, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, San Diego, CA, USA), 100 mg/ ml Streptomycin (Sigma Chemical Co., St Louis, MO, USA) 4 mM Lglutamine (Sigma) and 1 mM sodium pyruvate (Sigma). Human Burkitt lymphoma cell line Namalwa was maintained as suspension culture in RPMI 1640 medium supplemented with 10% FSB, 2 mM Lglutamine and 50 mg/ml Gentamycin (Sigma). The human U87CD4CXCR4 glioma cell line, was grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 mg/ml Gentamycin, 300 mg/ml G418 (Sigma) and 1 mg/ml puromycin (Sigma). HEK 293T cells stably expressing human TLR9 (HEK 293T/hTLR9) were developed by transfection of 6  105 cells per well, in a 6-well plate with 4 mg of the pUNO1-hTLR09a plasmid DNA (Invivogen), using Lipofectamine 2000 (Invitrogen). The pUNO1-hTLR09a vector expresses the human TLR09a (CD289) gene isoform 1 intronless open reading frame from the ATG to the stop codon (Invivogen, 6.3 kb). Fortyeight hours after transfection, cells were selected in DMEM containing 25 mg/ml blasticidin. Stable cell lines were propagated and maintained in complete DMEM supplemented with 25 mg/ml blasticidin. The cells were maintained at 37  C in a humidified atmosphere with 5% CO2. Adherent cells were harvested by trypsinization (0.25% trypsin, 0.02% EDTA) and seeded in 24-well plate at a cell density of 8e 10  104 cells per plate while Namalwa were seeded at a density of 5  105 cells/ml and incubated 24 h at 37  C for 24 h before transfection. The HEK 293T cells were transfected using lipofectamine 2000 transfectant agent (Invitrogen) with 0.5 mg of synthetic ssRNA and 0.5 mg synthetic dsRNA poly I:C (Sigma). The U87CD4CXCR4 cells were transfected with 0.5 mg of ssRNA. The immunostimulatory CpG ODN 2006 (TCGTCGTTTTGTCGTT TTGTCGTT) belonging to the class B was purchased from Invivogen. The immunostimulatory CpG motifs are underlined. HEK 293T/ hTLR9 and Nawalma cells were incubated with 2.5 mM ODN 2006 (Invivogen). 2.2. RNA isolation and RT-PCR analysis of TLR3, TLR7, TLR8, TLR9, IL-6, IL-8 and IL-10 gene expression Total RNA from transfected HEK 293T, HEK 293T/hTLR9, U87CD4CXCR4 and Namalwa cells were extracted using Trizol reagent (Invitrogen) following the manufacturer’s protocol. After treatment with RQ1 DNase (Promega) the extracted RNA was resuspended in 30 ml of nuclease-free water and stored at 80  C. RNA was quantified using NanoDrop ND-1000 spectrophotometric UV/Vis analysis (Nixor Biotech, Paris, France), and the integrity of total RNA was checked by agarose gel electrophoresis. Total RNA (1 mg) was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random hexamers, according to the manufacturer’s instructions. The PCR was set at 20 ml total volume, containing 2 ml of the cDNA sample, 0.625 units of Taq DNA polymerase (Thermo), 0.2 mM of each nucleotide, 75 mM of TriseHCl (pH 8.8 a 25  C), 20 mM (NH4)2 SO4, 0.01% TweenÒ 20, 1.5 mM MgCl2 and 10 pmol of each of the

corresponding primer set to detect the transcripts. Primers were designed according to the genbank sequences for human TLRs. Human TLR3 forward primer 50 -CCTGGTTTGTTAATTGGATTAACGA30 , reverse primer 50 -TGAGGTGGAGTGTTGCAAAGG-30 ; human TLR7 forward primer 50 -TTTACCTGGATGGAAACCAGCTA-30 , reverse primer 50 -TCAAGGCTGAGAAGCTGTAAGCTA-30 ; human TLR8 forward primer 50 -TTATGTGTTCCAGGAACTCAGAGAA-30 , reverse primer 50 -TAATACCCAAGTTGATAGTCGATAAGTTTG-30 ; human TLR9 forward primer 50 -GTGCCCCACTTCTCCATG-30 , reverse primer 50 GGCACAGTCATG ATGTTGTTG-30 ; human IL-6 forward primer 50 GGTACATCCTCGACGGCATCT-30 , reverse primer 50 -GTGCCTCTTT GCTGCTTTCAC-30 ; human IL-8 forward primer 50 -ATGACTTCCAAGCTGGCCGTGGCT-30 , reverse primer 50 -TCTCAGCCCTCTTCAAA ACTTCTC-30 ; human IL-10 forward primer 50 -ATGCCCCAAGCTGAGAACCA-30 , reverse primer 50 -CTGCTCCACGGCCTTGCTCTTGTT-30 . Control GAPDH mRNA was amplified with the forward primer GAPDH-F 50 -GAAGGTGAAGGTCGGAGTC-30 and the reverse primer GAPDH-R 50 -GAAGATGGTGATGGGATTTC-30 . PCR products were resolved in 1% or 2% agarose gels. 2.3. RNAi and ssRNA synthesis The oligonucleotide-directed production of small RNA transcripts with T7 RNA polymerase has been described [20]. For in vitro transcription, 45-nt DNA template oligonucleotides (PBS-as: 50 AAAGTGGCGCCCGAACAGGGACCTATAGTGAGTCGTATTACC-30 and PBS-s: 50 -AAGGTCCCTGTTCGGGCGCCACCTATAGTGAGTCGTATTA CC-30 ) and T7-si 50 -GGTATTACGACTCACTATAGG-30 oligonucleotide were designed to produce 21-nt siRNAs. The T7-si oligonucleotide was annealed to each of the other oligonucleotides in equimolar amounts in siRNA annealing buffer (10 mM TriseHCl, pH 8, 100 mM NaCl) by heating at 95  C for 2 min followed by slow cooling to room temperature over 1 h. The annealed oligonucleotides were transcribed in vitro using the T7-MEGAshortscript High Yield Transcription Kit (Ambion, Austin, TX, US) overnight at 37  C. DNase I was added to the transcription mixture which were incubated for 15 min at 37  C. The RNA was ethanol precipitated and resuspended in siRNA annealing buffer. Single-stranded RNAs were annealed to their complimentary RNA (antisense and sense strands) by heating to 95  C for 2 min and cooling to room temperature over 1 h. Double-stranded RNAs were purified on a 12% nondenaturing polyacrylamide gel run at 4  C and eluted overnight. Purified products were ethanol precipitated and resuspended in 20 ml siRNA annealing buffer. ssRNA (UACCAGGUAAUGUACCA CGACUUACGUCGUGUGUUUCUCUGGUUUGCU UCUAGUGUG) was in vitro transcribed by using the MEGAshortscript High Yield Transcription Kit (Ambion) according to the manufacturer’s specification. The resulting ssRNA was extracted with phenol/chloroform followed by ethanol precipitation. 2.4. One-step multiplex RT-PCR conditions Five gene-specific primers for IL-8, OAS2, IFNa or IFNß, and ISG56 were designed based on Primer 3 program (www-genome.wi.mit. edu) to amplify fragments of 373 bp (IFNa) or 370 bp (IFNß), 505 bp (OAS2), 137 bp (ISG56), 292 bp (IL-8); these were then evaluated separately and in a multiplex format. The primer pairs were: IFNa gene (NM_002173), IFNa-F: 50 -TTTCTCCTGCCTGAAGGACAG-30 and IFNa-R: 50 -TCTCATGATTTCTGCTCTGACA-30 or IFNb gene (NM_002176), 50 IFNb-F: 50 -CCTGTGGCAATTGAATGGGAGGC-30 and 30 IFNb-R: 50 -CCAGGCACAGTGACTGTCCTCCTT-30 ; OAS2 gene (NM_002535), OAS2-F: 50 -TGGCTCCTATGGACGGAAAACAGT-30 and OAS2-R: 50 -TGTTCCCAGGCATACACCGTA-30 ; ISG56 gene (NM_001548), ISG56-F: 50 -CTTGAGCCTCCTTGGGTTCG-30 and ISG56R: 50 -GCTGATATCTGGGTGCCTAAGG-30 ; IL-8 gene (NM_000584.2),

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IL-8F: 50 -ATGACTTCCAAGCTGGCCGTGGCT-30 and IL-8R: 50 TCTCAGCCCTCTTCAAAACTTCTC-30 . To test for possible repetitive sequences, the selected primers were aligned with the sequence databases at the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST) family of programs, to rule out non-specific binding to other human targets. One-tube RT-PCR was performed using the SuperScriptÔ One-Step RT-PCR Kit with PlatinumÒ Taq (Invitrogen). The 25 ml reaction mix consisted of: 12.5 ml of 2 reaction buffer, 1 ml RT/Platinum Taq Mix, 1 mg total RNA, 5 pmoles each GAPDH primers, 5 pmoles each IFNa or IFNb primers, 5 pmol each ISG56 primers, 20 pmol each OAS2 primers and 10 pmol each IL-8 primers, 2 mM MgCl2 and 0.8 mg/ml BSA. The optimized thermal profile included a reverse transcription step at 55  C for 30 min, followed by a denaturation step at 95  C for 3 min, the amplification was performed during 40 cycles including denaturation (94  C for 30 s), annealing (55  C for 30 s) and extension (68  C for 40 s) and was concluded by a final extension step at 68  C for 10 min and hold at 4  C. After PCR amplification, 5 ml of PCR products were mixed with loading dye and subjected to 2% agarose gel electrophoresis at 100 V for 45 min. After electrophoresis, the agarose gel was stained with 10% ethidium bromide solution for 10 min and visualized on an UV transilluminator. 3. Results 3.1. Expression of TLR3, TLR7, TLR8 and TLR9 in different cell lines The transcription levels of TLR3, TLR7, TLR8 and TLR9 were examined in HEK 293T, U87CD4CXCR4 and Namalwa cells. For this purpose a semi quantitative RT-PCR analysis of the mRNAs of TLR3, TLR7, TLR8 and TLR9 receptors from total RNA extracted from these human cell lines was performed. Results shown in Fig. 1 indicate

Fig. 2. Analysis of IL-6, IL-8 and IL-10 gene expression in HEK 293T/hTLR9 and Namalwa cell lines. Total RNA was isolated from HEK 293T/hTLR9 and Namalwa cells 24 h after incubation with 2.5 mM CpG ODN class B (OD2006) and IL-6, IL-8 and IL-10 mRNAs were individually examined by RT-PCR. Amplification products were resolved on 1 or 2% TAE agarose gels. Detection of the cellular GAPDH gene was used as internal RT-PCR control. (e), RT-PCR of total RNA extracted from untransfected cells.

that each of the tested cell lines expressed at different levels the different tested receptors involved in the recognition of nucleic acids. While the U87CD4CXCR4 cells constitutively expressed TLR3 (lane 2) and almost undetectable levels of TLR9 mRNA (lane 8), the Nawalwa cell line constitutively expresses the TLR7 receptor (lane 4) and TLR9 (lane 8). No expression of any of the tested receptors was observed when testing the HEK 293T cell line. These results imply that these cells should respond differently when they are treated with nucleic acids. 3.2. Induction of IL-8 gene expression in HEK 293T/hTLR9 cells by CpG DNA

Fig. 1. Analysis of TLR3, TLR7, TLR8 and TLR9 genes expression in HEK 293T, U87CD4CXCR4 and Namalwa cells. Total RNA was extracted from HEK 293T, U87CD4CRCX4 and Namalwa cell lines, and TLR3 (lane 2), TLR7 (lane 4), TLR8 (lane 6) and TLR9 (lane 8) mRNAs were individually examined by RT-PCR. GAPDH mRNA was used as internal reaction control. Genomic DNA of each cell line is included as a positive control of the PCR amplification reaction (lanes 1, 3, 5 and 7). A 100-bp DNA ladder was used as the DNA size marker (Lane M). Size, in bp, of interesting DNA marker fragments is shown on the right.

It has been shown that CpG ODNs are also capable of stimulating the release of cytokines IL-6, IL-8 and IL-10 in immune system cells [21,22]. To determine if the HEK 293T cells stably expressing human TLR9 (HEK 293T/hTLR9) could be used to analyze the immunogenicity of CpG ODN we test the effects of CpG ODNs on IL-6, IL-8 and IL-10 mRNA levels in Nawalma and HEK 293T/hTLR9 cells. These cell lines were incubated with the immunostimulatory class B CpG ODN 2006, and the mRNA levels of the mentioned cytokines were determined by semi quantitative RT-PCR (Fig. 2). We note that unlike the IL-6 and IL-10, IL-8 was induced in HEK 293T/hTLR9 cells stably expressing the human TLR9 when stimulated with a class B CpG ODN. For this reason, we included the detection of IL-8 gene in this multiplex RT-PCR method for analyzing the stimulation of the innate immune response by nucleic acids. These results demonstrate that a cell line that does not belong to the immune system also responds to exogenous nucleic acids when expressing a TLR that recognizes nucleic acids. Therefore, cells that do not belong to the immune system expressing TLR9 can be used to determine whether a particular CpG ODN is able to induce an innate immune response.

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3.3. Optimization of multiplex RT-PCR In order to rapidly and effectively evaluate the cellular response, induction of interferon and/or inflammatory cytokines, to the presence of exogenous nucleic acids we have developed a multiplex RT-PCR procedure. The multiplex RT-PCR assay was evaluated in terms of specificity and accuracy to ensure the efficiency of the technique. The developed assay allows the simultaneous analysis of expression of the IFNa or IFNb (data no shown), OAS2, ISG56 genes involved in the interferon response and the pro-inflammatory cytokine IL-8 gene, plus the GAPDH as internal reference gene. IFNa, ISG56, OAS2 genes are highly sensitive to the activation of the interferon response to nucleic acids and can be expressed in all cell types except for B lymphocytes, which do not induce the expression of ISG56 [23e26]. Optimization of the PCR conditions was essential for achieving reliable amplification. The multiplex RT-PCR conditions were tested and optimized in terms of annealing temperature (ranging from 50  C to 60  C) profile, extension temperature (ranging from 65  C to 72  C) and magnesium concentration (ranging from 1 to 5 mM). A large number of primer pairs were tested and their final optimal concentration (ranging from 0.04 to 0.8 mM) established empirically, varied considerably among the targeting gene to be amplified. Despite other possible RT-PCR conditions we defined the ones described under material and methods as optimum conditions for the efficient and reliable simultaneous detection of the five mentioned genes in a single reaction tube. Since it has been previously described that BSA, in concentrations up to 0.8 mg/ml increased the efficiency of the PCR much more than either DMSO or glycerol [27], the concentration of BSA was also optimized. A 0.8 mg/ ml BSA concentration in the reaction mix did not show any negative effect on any of the amplified loci (data not shown). The GAPDH message was used as internal control because its level in tissue culture cells is independent of the degree of confluence of the cell culture and is constant upon a variety of different treatments [28]. The only system in which GAPDH would not serve as an internal control is adipocyte differentiation where GAPDH is transcriptionally regulated [29]. The lack of GADPH amplification might indicate that the sample contains any RT-PCR inhibitory agent or low integrity of the extracted RNA. 3.4. Multiplex RT-PCR procedure to detect interferon and inflammatory stress response Once the PCR conditions were established and optimized, different samples were analyzed. Results of applying the multiplex RT-PCR in a single step to samples of total RNA extracted 24 h after transfection from various cell lines (HEK 293T, 293T/hTLR9, U87CD4CXCR4 and Namalwa) treated with various types of nucleic acids (ssRNA, dsRNA, siRNA and OD-2006) using primers specific for IFNa, ISG56, OAS2, IL-8 and GADPH genes are shown in Fig. 3. All treated cell lines yielded at least a specific amplicon of the expected size corresponding to one of the tested genes. The results also showed that the GAPDH gene was amplified in all samples, indicating the integrity of the RNA and the proficiency of the various reactions. Non unspecific PCR product was detected. As expected, untreated cells do not express genes involved in the activation of interferon response or IL-8 (Lanes 1, 4, 6 and 7). HEK 293T cells do not express TLR3, TLR7, TLR8 or TLR9 (Fig. 1). However, when they are stimulated with exogenous ssRNA (lane 2) or dsRNA (lane 3) induces interferon response which can be detected by the multiplex RT-PCR. Therefore this method can detect innate immune system activation in those cells lacking the TLRs involved in the recognition nucleic acids. U87CD4CXCR4 and HEK 293T cells have a different pattern of gene expression when stimulated with ssRNA

Fig. 3. Stimulation of interferon response and inflammatory stress detected in four human cell lines by exogenous nucleic acids. Lane 1, untransfected HEK 293T cell line (control). Lane 2, HEK 293T cell line transfected with 0.5 mg of ssRNA. Lane 3, HEK 293T cell line transfected with 0.5 mg of dsRNA. Lane 4, HEK 293T cell line stably expressing TLR9 (HEK 293T/hTLR9; control). Lane 5, HEK 293T/hTLR9 cell line, stimulated with 2.5 mM CpG ODN class B (OD2006). Lane 6, Namalwa cell line unstimulated (control). Lane 7, Namalwa cell line stimulated with 2.5 mM of CpG ODN class B (OD2006). Lane 8, U87CD4CXCR4 untransfected cell line (control). Lane 9, U87CD4CXCR4 cell line was transfected with 0.5 mg of ssRNA. Total RNA was isolated from each of the cells described 24 h after the transfection or stimulation. The three genes mediated interferon response (ISG56, IFNa, OAS2), the proinflammatory interleukin (IL-8), and the internal referent control of the reaction (GAPDH) were amplified simultaneously by RT-PCR in a single reaction mixture. All products were of the expected size. The transcription levels are determined by comparison with those obtained for the referent control gene. Lane M, 100-bp DNA ladder used as molecular size marker. Sizes, in bp, of selected DNA marker fragments are shown on the right.

(compare lanes 2 and 9) as occurs with HEK 293T/hTLR9 and Namalwa cells when they are stimulated with OD 2006 (compare lanes 5 and 7). All these data indicate that this multiplex RT-PCR is capable of detecting activation of the immune response due to stimulation with nucleic acids in different cell types. No discrepancies were observed when a monospecific RT-PCR for each gene, using only the specific primer pair under the same conditions, was performed showing that no interference occurred in the multiplex RT-PCR (data not shown). Basal and induced levels of IFNa or IFNb, ISG56, OAS2 and IL-8 may vary among different human cell lines, and even in a cell line depending on the growth conditions. Thus, it is critical to include a non-induced control that is treated exactly the same way as the tested samples (mock transfection control). 4. Discussion The achievements during the last 30 years in the development of nucleic acid tools as specific gene suppressors have led to a great increase in the number of attempts aimed to use the nucleic acids as inhibitors of gene expression or as therapeutic agents. However, immune stimulation and the induction of interferon and the inflammatory response by nucleic acids provide not only the potential for strong nonspecific effects with respect to target gene expression and function, but the result in significant toxicities when these molecules are administered in vivo. In addition, the development of an efficient strategy based on inhibitor nucleic acids requires the existence of highly sensitive tests that unequivocally confirm that the inhibitory or therapeutic effect is a direct action of the nucleic acid and no side effect due to nonspecific activation of an innate immune response release through TLR 3, 7, 8 and 9 or cytoplasmic receptors. Therefore it is recommended testing for interferon induction before attributing a particular response to a gene targeted nucleic acid. We have designed a onestep multiplex RT-PCR method to ensure whether synthetic CpG ODN, ssRNA and synthetic or plasmid-expressed siRNA induce interferon-related responses and/or proinflammatory cytokine release when introduced in the cell system of choice. Three classes of synthetic CpG ODNs; class A, B and C, activate human immune cells through TLR9 [21,22]. Recognition of synthetic CpG-ODNs through TLR9 has been shown to induce inflammatory response. Moreover, A- and C-class induce high levels of IFNa. In contrast, B-

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class ODNs preferentially induce inflammatory cytokines IL-6, IL-8 and IL-10 production but are relatively poor inducers of IFNa release. For this reason, we include the detection of the IL-8 gene expression in this method. The inclusion of the detection of this gene is a great advantage because it broadens the spectrum of cell types to which this method can be applied and to determine whether a particular CpG ODN is immunostimulatory without using immune system cells. Therefore, this multiplex RT-PCR might be applied to any cell treated with nucleic acids. Also, our procedure can be used to assess the effects of reagents (such as transfection reagents) and other procedural steps that may produce, enhance, or otherwise affect cellular interferon responses. The advantage of multiplex RT-PCR is that this assay comprises more than one primer set in a single reaction for detecting the presence of multiple target genes. This allows to reduce the amount of sample to be used that might be very limited and therefore of great value. It also allows reducing the manipulation minimizing the risk of contamination; it is less time-consuming and also reduced the cost of the analysis in comparison to other approaches based on monospecific gene amplification. To our knowledge this is the first report describing the use of a four primer pairs multiplex set for one step RT-PCR in a single tube for immune genes detection. We also observed here that expression of human TLR9 is correlated with CpG-DNA responsiveness in HEK 293T cells. Therefore, transfection of TLR9 into nonresponsive cells reconstitutes the immunostimulatory CpG-DNA response. 5. Conclusions The one-step multiplex RT-PCR method described for the analysis of the stimulation of the innate immune response by nucleic acids is advantageous because it is rapid, specific, sensitive, reproducible and cost effective allowing the simultaneous detection of five genes in a single reaction. This method allows evaluating different nucleic acid molecules, currently used for therapeutic or gene silencing approaches. Furthermore, as an internal reference control and to prevent false negative results it uses the detection of the gene GAPDH because its level of expression in culture cells is independent of the degree of cell confluence and it is constant under different treatments. Moreover, this method could be useful to determine the minimal concentration of nucleic acid able to induce interferon response. Therefore, this method could be used routinely in biological and clinical research laboratories to test activation of interferon and/or inflammatory stress related pathways of cells by the introduction of nucleic acids for knockdown gene or therapeutic applications. Acknowledgments We would like to thank Dr. Sánchez-Luque for helpful comments and Vicente Augustin-Vacas for his excellent technical assistance. This work was supported by grants BFU2009-08137 and BFU201231213 from the Spanish Ministerio de Ciencia e Innovación, and grant 36472/05 from FIPSE to A. B-H. J.A.R-D. was funded by grant 36472/ 05 from FIPSE. Work at our laboratory is partially supported by FEDER funds from the EU. References [1] Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004;3:318e29.

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Detection of immune response activation by exogenous nucleic acids by a multiplex RT-PCR method.

Transfection of mammalian cells or in vivo administration of nucleic acids can induce inflammatory cytokines and/or interferon response, which could s...
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