A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5′ RACE and a molecular beacon probe

Lasham, Annette; Herbert, Mike; Coppieters, Natacha; Patel, Rachna; Feng, Sheryl; Eszes, M; Cao, Helen; Reid, Glen

doi:10.1093/nar/gkp1076

Cited by 19 publications

(19 citation statements)

References 40 publications

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“…5′ RLM-RACE was performed using the GeneRacer Kit (Invitrogen) with some modification as previously described. 49 Briefly, 100 ng total RNA was directly ligated to 250 ng GeneRacer RNAOligo with T4 ligase. After phenol/chloroform extraction and ethanol precipitation, cDNA was synthesized using random primers.…”

Section: Red Blood Cell Hemolysis Assaymentioning

confidence: 99%

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

et al. 2011

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The application of small interfering RNA (siRNA) for cancer treatment is a promising strategy currently being explored in early phase clinical trials. However, efficient systemic delivery limits clinical implementation. We developed and tested a novel delivery system comprised of (i) an internalizing streptavidin-conjugated monoclonal antibody (mAb-SA) directed against CD22 and (ii) a biotinylated diblock copolymer containing both a positively charged siRNA condensing block and a pH-responsive block to facilitate endosome release. The modular design of the carrier facilitates the exchange of different targeting moieties and siRNAs to permit its usage in a variety of tumor types. The polymer was synthesized using the reversible addition fragmentation chain transfer (RAFT) technique and formed micelles capable of binding siRNA and mAb-SA. A hemolysis assay confirmed the predicted membrane destabilizing activity of the polymer under acidic conditions typical of the endosomal compartment. Enhanced siRNA uptake was demonstrated in DoHH2 lymphoma and transduced HeLa-R cells expressing CD22 but not in CD22 negative HeLa-R cells. Gene knockdown was significantly improved with CD22-targeted vs. nontargeted polymeric micelles. Treatment of DoHH2 cells with CD22-targeted polymeric micelles containing 15 nmol/l siRNA produced 70% reduction of gene expression. This CD22-targeted polymer carrier may be useful for siRNA delivery to lymphoma cells.

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Section: Red Blood Cell Hemolysis Assaymentioning

confidence: 99%

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

et al. 2011

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“…The first step involved the development of a method for quantifying full-length mRNA and one of the truncated transcripts, and for this we selected Molecular Beacon Rapid Amplification of cDNA Ends (MBRACE) [11] (outlined in Figure 2A). In this assay, cytoplasmic RNA is first treated with a phosphatase to prevent adapter ligation to uncapped ends.…”

Section: Resultsmentioning

confidence: 99%

“…To address this we developed an inducible line of erythroid cells which were used to monitor the cytoplasmic appearance of full-length normal (WT-hβG) and PTC-containing (PTC-hβG) human β-globin mRNA after treating cells with doxycycline to induce transcription of their respective genes. Changes in full-length mRNA and one of the 5′-truncated RNAs were determined using a modification of Molecular Beacon Rapid Amplification of cDNA Ends (MBRACE) [11], a qRT-PCR-based assay for quantifying products after ligation of a common primer to uncapped 5′ ends.…”

Section: Introductionmentioning

confidence: 99%

SMG6 Cleavage Generates Metastable Decay Intermediates from Nonsense-Containing β-Globin mRNA

2013

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mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human β-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5′-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA missing 169 nucleotides from the 5′ end. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5′-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process full-length nonsense-containing β-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.

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“…For gapmers or siRNAs, a technique called rapid amplification of 5′-cDNA ends (5′-RACE) can be used to verify that the targeted transcript is being cleaved at the predicted site [127–130]. While valuable, it is important to note that 5′-RACE merely detects cleavage, it is not quantitative and is not an indication of efficiency.…”

Section: Control Experiments: You Can Never Have Too Manymentioning

confidence: 99%

Silencing disease genes in the laboratory and the clinic

Watts

Corey

2011

The Journal of Pathology

380

309

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Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology.

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A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5′ RACE and a molecular beacon probe

Cited by 19 publications

References 40 publications

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

Anti-CD22 Antibody Targeting of pH-responsive Micelles Enhances Small Interfering RNA Delivery and Gene Silencing in Lymphoma Cells

SMG6 Cleavage Generates Metastable Decay Intermediates from Nonsense-Containing β-Globin mRNA

Silencing disease genes in the laboratory and the clinic

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