Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments

Gyöngyi Munkácsy, Zsófia Sztupinszki, Péter Herman, Bence Bán, Zsófia Pénzváltó, Nóra Szarvas, B. Györffy

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.

Original languageEnglish
Pages (from-to)e366
JournalMolecular Therapy - Nucleic Acids
Volume5
DOIs
Publication statusPublished - 2016

Fingerprint

RNA Interference
Gene Silencing
Small Interfering RNA
Genes
Transfection
Cell Line
Gene Expression Profiling
Nonparametric Statistics
Oligonucleotide Array Sequence Analysis
Publications
Down-Regulation
Western Blotting
Polymerase Chain Reaction

Keywords

  • cancer
  • cell line
  • gene silencing
  • microarrays
  • RNA interference
  • transfection

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

Cite this

Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments. / Munkácsy, Gyöngyi; Sztupinszki, Zsófia; Herman, Péter; Bán, Bence; Pénzváltó, Zsófia; Szarvas, Nóra; Györffy, B.

In: Molecular Therapy - Nucleic Acids, Vol. 5, 2016, p. e366.

Research output: Contribution to journalArticle

Munkácsy, Gyöngyi ; Sztupinszki, Zsófia ; Herman, Péter ; Bán, Bence ; Pénzváltó, Zsófia ; Szarvas, Nóra ; Györffy, B. / Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments. In: Molecular Therapy - Nucleic Acids. 2016 ; Vol. 5. pp. e366.
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