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IFNr qRT-Primers - Detection of the IFN Response Double strand RNA (dsRNA) is a molecular pattern associated with viral infection. It is recognized by the innate immune system leading to the production of type I interferons and the subsequent induction of a variety of antiviral genes. Recent reports indicate that certain siRNAs and shRNAs can activate the IFN pathway, potentially leading to severe side effects. To determine whether a given siRNA or shRNA is immunostimulatory, InvivoGen has developed a set of primers that detect the expression of human genes involved in the IFN response by real-time quantitative PCR using the SYBR ® Green detection. ➥ The IFNr qRT-Primers provide highly specific and sensitive results in real-time quantitative PCR. ➥ Each IFNr qRT-Primer Pair is carefully designed and tested. ➥ The size of the amplified fragments varies from 80 to 280 bp. ➥ The IFNr qRT-Primers allow to perform 100 x 50 μl reactions (in a 96-well plate) or 250 x 20 μl reactions (in a 384-well plate). Description The IFNr qRT-Primers are a set of primer pairs designed to measure the mRNA expression of 5 genes induced by IFNa: - IFNb - OAS1 - MX1 (also known as MxA) - G1P2 (also known as ISG15) - IFIT1 (also known as CDKL2 or P56), and the housekeeping gene GAPDH as a control. The IFNr qRT-Primers allow one-step or two-step qRT-PCR in combination with SYBR ® Green I dye. Interferon Response: An Off-Target Effect The RNAi reagents, siRNA and shRNA, can be potent inducers of interferons (IFNs) and inflammatory cytokines both in vivo and in vitro 1-3 due to their recognition by cellular receptors of foreign RNA and DNA. Much of the IFN response is caused by the activation of the dsRNA-dependent protein kinase R (PKR), that recognizes long double-stranded (ds)RNA, leading to a global inhibition of protein synthesis. siRNAs that are shorter than 30 bp can evade PKR activation, but are recognized by other receptors, such as the Toll-like receptors (TLRs). TLR3, which ligand is dsRNA, and TLR7 and TLR8, that sense single-stranded RNA, have been implicated in the recognition of siRNAs 1-4 . Recognition by TLR7 and TLR8 appears to sequence-dependent as several immunostimulatory sequences have been identified. These sequences are characterized by a high content of guanosine and uridine residues 1, 5 . They have been integrated in InvivoGen’s siRNA Wizard algorithm and are systematically excluded to limit the IFN response. The IFN response induced by shRNAs results mainly from the DNA vector that contains non-methylated CpG sequences that are recognized by TLR9. InvivoGen has designed a CpG-free vector, pCpGfree-siRNA, to overcome this problem (see page 137). Contents and Storage The IFNr qRT-Primers contain 10 μM of each primer. The 5’ sense primer and the 3’ antisense primer are provided in separate vials. Products are lyophilized and shipped at room temperature. PRODUCT QTY CAT. CODE IFNr qRT-Primers 1 kit rts-hifnr 1. Hornung V. et al., 2005. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 11(3):263-70. 2. Sioud M., 2005. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol. 348(5):1079-90. 3. Agrawal S. & Kandimalla ER. 2004. Antisense and siRNA as agonists of Toll-like receptors. Nat. Biotechnol. 22: 1533-1537. 4. Kariko K. et al., 2004. Exogenous siRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3. Cells Tissues Organs. 177(3):132-8. 5. Judge AD. et al., 2005. Sequencedependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 23(4):457-62. www.invivogen.com/interferon-response 147 RNA INTERFERENCE 7
RNA INTERFERENCE 7 Ready-Made psiRNA - Plasmids Expressing Functional shRNAs Ready-made psiRNA is a new family of plasmids expressing a growing list of shRNAs which functionality has been described in the literature or validated in house. Ready-made psiRNA plasmids eliminate the need to design and clone several siRNA sequences before identifying an effective one. They express shRNAs that silence the expression of a target gene by >70%. Features and Benefits Ready-made psiRNAs are psiRNA-h7SKGFPzeo-derived plasmids. They feature the human 7SK RNA Pol III promoter that generates high amounts of short hairpin RNAs. They also feature the GFP::Zeo fusion gene which confers both reporter and antibiotic resistance activities making these plasmids very useful for the following applications: • Monitor transfection efficiency • Standardize gene silencing efficiency • Select clones that stably express a validated siRNA The silencing efficiency of each Ready-made psiRNA plasmid has been tested using the psiTEST system (see page 146). The genes or fragments of the genes targeted by the Ready-made psiRNA have been fused to the SEAP reporter gene within the psiTEST plasmid. Silencing efficiencies have been confirmed by the absence of SEAP activity after cotransfecting HEK293 cells with each recombinant psiTEST and corresponding Readymade psiRNA. Contents and Storage Ready-made psiRNA plasmids are available alone or in a kit. Ready-made psiRNA plasmids are provided as 20 μg of lyophilized DNA. Each Ready-made psiRNA kit contains the following components: - 20 μg of a Ready-made psiRNA plasmid - 20 μg of a control psiRNA plasmid (psiRNA-Luc) - 1 vial of LyoComp GT116 - 4 pouches of Fast-Media ® Zeo Products are shipped at room temperature. Store at -20°C. PRODUCT QTY CAT. CODE* Ready-made psiRNA plasmid 20 μg psirna42- Ready-made psiRNA kit 1 kit ksirna42- *See tables 148 www.invivogen.com/readymade-psirna GENE NAME/ALIASES SPECIES CAT. CODE (Plasmid)** A20 / TNFAIP3 Human, mouse psirna42-(h/m)a20 ASC / CARD5 Human, mouse psirna42-(h/m)asc AIM2 / IFI210 Human, mouse psirna42-(h/m)aim2 ATF3 Human psirna42-hatf3 ATG5 Human, mouse psirna42-(h/m)atg5 BAF47 / SMARCB1 Human psirna42-hbaf47 BP1 NEW Human psirna42-hbpi BCL2 Human psirna42-hbcl2 BCL3 Human, mouse psirna42-(h/m)bcl3 BCL10 / CLAP Human, mouse psirna42-(h/m)bcl10 Beclin-1 Human, mouse psirna42-(h/m)beclin CARD8 / Cardinal Human psirna42-hcard8 CARD9 Human, mouse psirna42-(h/m)card9 Caspase 1 / ICE Human, mouse psirna42-(h/m)casp1 Caspase 8 / FLICE Human, mouse psirna42-(h/m)casp8 CD14 Human, mouse psirna42-(h/m)cd14 CD36 Human, mouse psirna42-(h/m)cd36 CD44 Human, mouse psirna42-(h/m)cd44 CLEC9A Human, mouse psirna42-(h/m)clec9a CXCL16 NEW Human, mouse psirna42-(h/m)cxcl16 CXCR4 / CD184 Human psirna42-hcxcr4 CYLD Human psirna42-hcyld DAI / ZBP1 Human psirna42-hdai DAK Human, mouse psirna42-(h/m)dak DC-SIGN / CD209 Human, mouse psirna42-(h/m)dcsign DDX3 / DDX3X Human psirna42-hddx3x Dectin-1 Human, mouse psirna42-(h/m)dectin1 DNMT1 Human psirna42-hdnmt1 DNMT2 Human psirna42-hdnmt2 DNMT3A Human psirna42-hdnmt3a DNMT3B Human psirna42-hdnmt3b DNMT3L Human psirna42-hdnmt3l DUBA / OTUD5 Mouse psirna42-mduba FADD / MORT1 Human, mouse psirna42-(h/m)fadd FLII / Fliih Human psirna42-hflii GM-CSF Human, mouse psirna42-(h/m)gmcsf HDAC1 Human psirna42-hhdac1 HDAC2 Human psirna42- hhdac2 HDAC3 Human psirna42-hhdac3 HDAC6 Human psirna42-hhdac6 HDAC8 Human psirna42-hhdac8 HPRT Human psirna42-hhprt IFI16 / PYHIN2 NEW Human psirna42-hifi16 IFNAR1 Human psirna42-hifnar1 ** For the kit catalog code, replace psirna42 by ksirna42
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INNOVATION WITHIN REACH InvivoGen
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CONTENTS AT A GLANCE 1 CELL CULTURE
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4 CYTOKINE SIGNALING TABLE of CONTE
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Mycoplasma Detection & Elimination
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PlasmoTest Contents - HEK-Blue -2
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Primocin - The Antimicrobial Shiel
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SEAP Reporter Gene System Secreted
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HEK-Blue Detection - Real-Time Det
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Blasticidin S Description Blasticid
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LyoVec New formulation LyoVec is
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Poly-Arginine-HA Tagged Reprogrammi
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LENTI-Smart - Lentiviral Vector Pr
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Lentiviral Vector Production and Ce
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Antibody generation using pFUSE-CHI
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Engineering Fc regions for altered
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pBROAD & pWHERE - Optimized Vector
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pSELECT-Tag - Expression of a GFP-,
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pVIVO - Expression of Two Genes of
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pORF Description pORF plasmids feat
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Prom A-List - Human and Rodent Pro
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PROMOTER GENE TISSUE DISTRIBUTION S
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ChemiComp E. coli ChemiComp E. coli
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Fast-Media ® Preparation 1. Empty
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RIG-I-like Receptors 54 RLR & RLR-R
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Toll-Like Receptors Toll-Like Recep
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www.invivogen.com/innate-immunity 5
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IPAF and NAIP5 IPAF (NLRC4, CLAN/CA
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C-Type Lectin Receptors C-type lect
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pUNO pUNO-HA Toll-Like Receptors (T
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GENE NAME/ALIASES DESCRIPTION CAT.
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pDUO - Gene Associations Features a
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pZERO-TLR & pDeNy - Dominant Negati
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293/TLR & 293/NOD Clones 293/TLR Cl
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Response of HEK-Blue hTLR2 cells t
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Ramos-Blue Cells - NF-kB/AP-1 Repo
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MEF Cells C3H/TLR4mut & C3H/WT MEFs
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Antibodies for Detection ANTIBODY D
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- NF-kB binding site: Nuclear facto
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PRODUCT ORIGIN/DESCRIPTION TLR2 Ago
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PRODUCT ORIGIN/DESCRIPTION TLR9 Ago
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PRODUCT ORIGIN/DESCRIPTION Dectin-1
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LPS-PG (Porphyromonas gingivalis) R
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• FLA-ST ultrapure was purified b
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1. Zhao H. et al., 2004. Contributi
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1. Hornung V. et al., 2006. 5'-Trip
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TLR/NOD Ligand Screening InvivoGen
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Signal Transduction Inhibitors Invi
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Pepinh-MYD - MyD88 inhibitory pepti
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Page 100 and 101: Nigericin - NLRP3 Inflammasome Indu
Page 102 and 103: Autophagy is a pathway by which cyt
Page 104 and 105: 4 CYTOKINE SIGNALING Cytokine Genes
Page 106 and 107: Blue Cytokine Reporter Cells Blue
Page 108 and 109: HEK-Blue IFN-a/b Cells - Human Typ
Page 110 and 111: HEK-Blue IL-6 Cells - IL6 Reporter
Page 112 and 113: HEK-Blue IL-1b Cells - IL-1b Repor
Page 114 and 115: HEK-Blue IL-33/IL-1b Cells - IL-33
Page 116 and 117: HEK-Blue CD40L Cells - CD40L Repor
Page 118 and 119: 5 ANTIBODIES & VACCINATION Antibodi
Page 120 and 121: Antibody generation using pFUSE-CHI
Page 122 and 123: Immunoglobulin A The mucosal surfac
Page 124 and 125: IgA Detection and Quantification In
Page 126 and 127: IgG Antibodies InvivoGen offers a s
Page 128 and 129: Mediators? 1. Leroux-Roels G., 2010
Page 130 and 131: Poly(I:C) Synthetic double-stranded
Page 132 and 133: pVAC - Induction of Neutralizing An
Page 134 and 135: CpG-Free DNA CpGs and the Immune Re
Page 136 and 137: pCpGfree-basic & pCpGfree-promoter
Page 138 and 139: pCpGfree-siRNA & pCpGfree-siRNADUO
Page 140 and 141: Custom-Made CpG-Free Genes InvivoGe
Page 143 and 144: RNA INTERFERENCE 7 psiRNA System I
Page 145 and 146: RNA INTERFERENCE 7 psiRNA-h7SK - 7S
Page 147: RNA INTERFERENCE 7 psiTEST System
Page 151 and 152: RNA INTERFERENCE 7 Custom-Made psiR
Page 153 and 154: INHIBITORS 8 Inhibitors PRODUCT DES
Page 155 and 156: INHIBITORS 8 Pattern Recognition Re
Page 157 and 158: INHIBITORS 8 Triptolide - NF-kB Inh
Page 159 and 160: INHIBITORS 8 Heat Shock Protein Inh
Page 161 and 162: 9 CUSTOM SERVICES TLR/NOD Ligand Sc
Page 163 and 164: CUSTOM SERVICES 9 Custom Cloning :
Page 165 and 166: CUSTOM SERVICES 9 Prices 164 TERMS
Page 167 and 168: 166 PRODUCT (QUANTITY) CAT. CODE PA
Page 173 and 174: A 17-AAG 17-AAGH2 17-AEP-GA 2-Amino
Page 175 and 176: - ODN 4084-F - ODN INH-1 - ODN INH-
Page 177 and 178: ARGENTINA Genbiotech S.R.L. Tel: +5
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