The template for dsRNA
synthesis was obtained by polymerase chain reaction (PCR) performed in a reaction volume of 50 [micro]L comprising 36.5 [micro]L PCR-grade water, 5 ui 10X Taq buffer, 1.0 [micro]L (10 mM) dNTP mix, 1.0 [micro]L (10 mM) each of forward and reverse primers (Table 1), 2.5 [micro]L 1:50 diluted plasmid clone as template, and 1.0 [micro]L Taq polymerase (3 U/[micro]L) (Fermentas Life Sciences, USA).
Properties of representative chemically identified adjuvants Category Adjuvant Receptor Adaptor Lipid MPL TLR4 MyD88/TICAM-1 Lipopeptide TLR2 (+TLR1 or TLR6) MyD88 Oil, squalane Undefined Undefined Protein Flagellin TLR5 MyD88 Cholera toxin B GM1 receptor G proteins Versican TLR2 MyD88 Nucleic acid Virus dsRNA
TLR3, MDA5 TICAM-1 /MAVS Poly I:C TLR3, MDA5 TICAM-1 /MAVS CpG DNA TLR9 MyD88 STING ligand cGAS etc.
We have previously demonstrated that the viral dsRNA
synthetic mimetic poly(I:C) upregulates the secretion of proinflammatory cytokines and prolabour mediators in the human myometrium .
In RT-2 reaction, denatured dsRNA
was then added to the reaction mixture consisting of 10 l of the 5X reaction buffer, 1 l of 10 mM deoxynucleoside triphosphate (10mM dNTP) mixture, 2 l of 25 mM MgSO4, 1 l of MuLV Reverse Transcriptase enzyme (5U/ul), 1 l of Taq Polymerase (5U/ul) and 3 l of dimethyl sulfoxide in a final volume of 50 l and then preincubated at 480C for 45 min followed by 35 cycles of 940C for 1 min., 520C for 2 min., and 680C for 3 min.
The authors suggested that when the alpha-helix CPP is tilted, it can fit into the major groove of the dsRNA
via hydrogen bonds and van der Waals interactions [27-29].
RNA interference (RNAi) is a cellular process whereby double-stranded RNA (dsRNA
) leads to posttranscriptional gene silencing through base-pairing interactions and is found in many eukaryotic systems, including plants, fungi, invertebrates, and mammals [1-4].
Different types of nucleic acid have their unique TLR, with viral double-stranded RNA (dsRNA
) being recognised by TLR3, single-stranded RNA (ssRNA) being recognised by TLR7 and TLR8, and DNA containing unmethylated CG dinucleotides (whether from bacteria, viruses, or synthetic oligodeoxynucleotides, referred to as ODNs) being recognised by TLR9 [1,10,11].
coli strain HT115 (DE3) bacteria carrying daf-16 dsRNA
. The RNAi plates were prepared with NGM containing 1mM isopropyl [beta]-D-1-thiogalactopyranoside (IPTG) and 50 [micro]g/mL ampicillin.
Dillon, "Gene silencing in phlebotomine sand flies: xanthine dehydrogenase knock down by dsRNA
microinjections," Insect Biochemistry and Molecular Biology, vol.
1, part I), pri-miRNAs are processed in the nucleus by the Drosha and the double stranded RNA (dsRNA
)-binding protein of DiGeorge Syndrome Critical Region Gene 8 (DGCR8) RNase-ribonucleoprotein complex into approximately 70-100 bp long precursor miRNAs (premiRNAs).
complementary DNA; CNN, centrosomin: CPC, chromosomal passenger complex; DAPI, 4',6'-diamidino-2-phenylindole: DMSO, dimethyl sulfoxide: dsRNA
, double-stranded RNA; FBS, fetal bovine serum: FRET.
Through these proteins, the stem-loop hairpin transcripts of endogenous genes are processed to generate miRNAs or long dsRNA
precursors (either exogenous or endogenous) that are cut to siRNAs.