Effect of biological matrix and sample preparation on qPCR quantitation of siRNA drugs in animal tissues

Seitzer, Jessica; Zhang, Hangchun; Koser, Martin L.; Pei, Yi; Abrams, Marc

doi:10.1016/j.vascn.2010.09.005

Cited by 14 publications

(12 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Following systemic injection of siRNA particles in vivo, the total levels of the siRNA payload in blood and its distribution in different organs can be quantitatively addressed by stem-loop reverse transcription quantitative polymerase chain reaction (stem-loop RT-qPCR) (Stratford et al 2008;Abrams et al 2010;Seitzer et al 2011). This method is applied to quantitatively measure siRNA in any biological sample in a high-throughput manner.…”

Section: Approaches To Evaluate Sirna Overall Tissue Distributionmentioning

confidence: 99%

“…This is the reason that total siRNA in tissues does not always generally agree with efficacy Seitzer et al 2011). Wei et al (2011 demonstrated that target mRNA silencing in LNP-siRNA-treated livers correlated with the amount of siRNA bound to Ago2 but not with the siRNA measured in total liver lysates.…”

Section: Analyses Of Sirna Endosomal Escape and Riscloaded Sirnamentioning

confidence: 99%

See 1 more Smart Citation

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

Shu¹,

Abrams²

2013

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

Small interfering RNA (siRNA) therapeutics have advanced from bench to clinical trials in recent years, along with new tools developed to enable detection of siRNA delivered at the organ, cell, and subcellular levels. Preclinical models of siRNA delivery have benefitted from methodologies such as stem-loop quantitative polymerase chain reaction, histological in situ immunofluorescent staining, endosomal escape assay, and RNA-induced silencing complex loading assay. These technologies have accelerated the detection and optimization of siRNA platforms to overcome the challenges associated with delivering therapeutic oligonucleotides to the cytosol of specific target cells. This review focuses on the methodologies and their application in the biodistribution of siRNA delivered by lipid nanoparticles.

show abstract

Section: Approaches To Evaluate Sirna Overall Tissue Distributionmentioning

confidence: 99%

Section: Analyses Of Sirna Endosomal Escape and Riscloaded Sirnamentioning

confidence: 99%

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

Shu¹,

Abrams²

2013

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

show abstract

“…RPMI 1640 medium, EMEM medium, fetal bovine serum (FBS), 0.25% trypsin-EDTA solution, penicillin-streptomycin (P/S) and lymphocyte separation medium (LSM) were purchased from WISENT Bioproduct Inc (Montréal, Qc, Canada). siRNA-SSB (GenBank accession number NM_009278) with the oligonucleotide sequence: antisense 5ʹ-uuacauuaaagucuguuguTT-3ʹ; and sense 5ʹ-acaacagacuuuaauguaaTT-3ʹ (as mentioned by Abrams et al, 24 and Seitzer et al) 25 with a 2ʹ-O-Me-rA (rC, rG, rU) modification, was purchased from Alpha DNA S.E.N.C. (Montréal, Qc, Canada).…”

Section: Methodsmentioning

confidence: 99%

<p>Evidence Supporting the Safety of Pegylated Diethylaminoethyl-Chitosan Polymer as a Nanovector for Gene Therapy Applications</p>

Rondon¹,

Benabdoun²,

Vallières³

et al. 2020

IJN

View full text Add to dashboard Cite

Purpose: Diethylaminoethyl-chitosan (DEAE-CH) is a derivative with excellent potential as a delivery vector for gene therapy applications. The aim of this study is to evaluate its toxicological profile for potential future clinical applications. Methods: An endotoxin-free chitosan (CH) modified with DEAE, folic acid (FA) and polyethylene glycol (PEG) was used to complex small interfering RNA (siRNA) and form nanoparticles (DEAE 12-CH-PEG-FA 2 /siRNA). Based on the guidelines from the International Organization for Standardization (ISO), the American Society for Testing and Materials (ASTM), and the Nanotechnology Characterization Laboratory (NCL), we evaluated the effects of the interaction between these nanoparticles and blood components. In vitro screening assays such as hemolysis, hemagglutination, complement activation, platelet aggregation, coagulation times, cytokine production, and reactive species, such as nitric oxide (NO) and reactive oxygen species (ROS), were performed on erythrocytes, plasma, platelets, peripheral blood mononuclear cells (PBMC) and Raw 264.7 macrophages. Moreover, MTS and LDH assays on Raw 264.7 macrophages, PBMC and MG-63 cells were performed. Results: Our results show that a targeted theoretical plasma concentration (TPC) of DEAE 12-CH-PEG-FA 2 /siRNA nanoparticles falls within the guidelines' thresholds: <1% hemolysis, 2.9% platelet aggregation, no complement activation, and no effect on coagulation times. ROS and NO production levels were comparable to controls. Cytokine secretion (TNF-α, IL-6, IL-4, and IL-10) was not affected by nanoparticles except for IL-1β and IL-8. Nanoparticles showed a slight agglutination. Cell viability was >70% for TPC in all cell types, although LDH levels were statistically significant in Raw 264.7 macrophages and PBMC after 24 and 48 h of incubation. Conclusion: These DEAE 12-CH-PEG-FA 2 /siRNA nanoparticles fulfill the existing ISO, ASTM and NCL guidelines' threshold criteria, and their low toxicity and blood biocompatibility warrant further investigation for potential clinical applications.

show abstract

“…A multitude of RT-qPCR-based assays have been developed to quantitate siRNAs and microRNAs in biological matrices, including primer-extension qPCR [20], stem-loop PCR [21,22], universal probe library RT-PCR [23], and others [24][25][26]. Quantitative PCR is a sensitive method with the ability to measure pg/mL concentrations of siRNAs with relative ease using commercially available reagents.…”

Section: Current Analytical Methodologiesmentioning

confidence: 99%

Mass Spectrometry of siRNA

Cancilla

Flanagan²

2013

Mass Spectrometry for Drug Discovery and Drug Development

View full text Add to dashboard Cite

Effect of biological matrix and sample preparation on qPCR quantitation of siRNA drugs in animal tissues

Cited by 14 publications

References 18 publications

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

<p>Evidence Supporting the Safety of Pegylated Diethylaminoethyl-Chitosan Polymer as a Nanovector for Gene Therapy Applications</p>

Mass Spectrometry of siRNA

Contact Info

Product

Resources

About