title: Decoding pooled RNAi screens by means of barcode tiling arrays.
abstract: BACKGROUND
  RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool.
  
  
  RESULTS
  We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach.
  
  
  CONCLUSIONS
  In summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.
authors.affiliation: Department for Functional Genome Analysis, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany. m.boettcher@dkfz.de
authors.forename: Michael
authors.initials: M
authors.lastname: Boettcher
authors.forename: Johannes
authors.initials: J
authors.lastname: Fredebohm
authors.forename: Amin Moghaddas
authors.initials: AM
authors.lastname: Gholami
authors.forename: Yafit
authors.initials: Y
authors.lastname: Hachmo
authors.forename: Iris
authors.initials: I
authors.lastname: Dotan
authors.forename: Dan
authors.initials: D
authors.lastname: Canaani
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Hoheisel
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D006801:Humans; D015341:Nucleic Acid Probes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D034622:RNA Interference; D012334:RNA, Neoplasm / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051603:Recoverin; D015203:Reproducibility of Results
chemical_list: D015341:Nucleic Acid Probes; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D051603:Recoverin