title: Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells.
abstract: BACKGROUND
  Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell.
  
  
  METHODS
  In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis.
  
  
  RESULTS
  We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles.
  
  
  CONCLUSIONS
  The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; School of Life and Health Science, Aston University, Birmingham, United Kingdom.
authors.forename: Meesbah
authors.initials: M
authors.lastname: Jiwaji
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; Division of Biomedical Engineering, School of Engineering, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Mairi E
authors.initials: ME
authors.lastname: Sandison
authors.affiliation: Division of Biomedical Engineering, School of Engineering, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Julien
authors.initials: J
authors.lastname: Reboud
authors.affiliation: Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom.
authors.forename: Ross
authors.initials: R
authors.lastname: Stevenson
authors.affiliation: School of Computing Science, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Rónán
authors.initials: R
authors.lastname: Daly
authors.affiliation: Institute of Cancer Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Gráinne
authors.initials: G
authors.lastname: Barkess
authors.affiliation: Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Faulds
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; Systems Biology Ireland and the Conway Institute, University College Dublin, Belfield, Dublin, Ireland.
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.affiliation: Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United Kingdom.
authors.forename: Duncan
authors.initials: D
authors.lastname: Graham
authors.affiliation: School of Computing Science, University of Glasgow, Glasgow, United Kingdom; Department of Statistical Science, University College London, London, United Kingdom.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Girolami
authors.affiliation: Division of Biomedical Engineering, School of Engineering, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Cooper
authors.affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; School of Life and Health Science, Aston University, Birmingham, United Kingdom.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Pitt
mesh_terms: D004277:DNA, Single-Stranded / Q000494:pharmacology; D055785:Gene Knockdown Techniques / Q000379:methods*; D006046:Gold; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection
chemical_list: D004277:DNA, Single-Stranded; C491053:MT2A protein, human; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D006046:Gold; D008668:Metallothionein