Decoding pooled RNAi screens by means of barcode tiling arrays

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype

Introduction: Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. Methods: We describe a sub-population expressing the stem-like marker CD44+/CD24-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44+/CD24low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. Results: Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. Conclusion: In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A

Bioinformatic, structural, and functional analyses support release factor-like MTRF1 as a protein able to decode nonstandard stop codons beginning with adenine in vertebrate mitochondria

Vertebrate mitochondria use stop codons UAA and UAG decoded by the release factor (RF) MTRF1L and two reassigned arginine codons, AGA and AGG. A second highly conserved RF-like factor, MTRF1, which evolved from a gene duplication of an ancestral mitochondrial RF1 and not a RF2, is a good candidate for recognizing the nonstandard codons. MTRF1 differs from other RFs by having insertions in the two external loops important for stop codon recognition (tip of helix α5 and recognition loop) and by having key substitutions that are involved in stop codon interactions in eubacterial RF/ribosome structures. These changes may allow recognition of the larger purine base in the first position of AGA/G and, uniquely for RFs, only of G at position 2. In contrast, residues that support A and G recognition in the third position in RF1 are conserved as would be required for recognition of AGA and AGG. Since an assay with vertebrate mitochondrial ribosomes has not been established, we modified Escherichia coli RF1 at the helix α5 and recognition loop regions to mimic MTRF1. There was loss of peptidyl-tRNA hydrolysis activity with standard stop codons beginning with U (e.g., UAG), but a gain of activity with codons beginning with A (AAG in particular). A lower level of activity with AGA could be enhanced by solvent modification. These observations imply that MTRF1 has the characteristics to recognize A as the first base of a stop codon as would be required to decode the nonstandard codons AGA and AGG

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

<div><p>Objectives</p><p>Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.</p><p>Methods</p><p>Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K₂EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).</p><p>Results</p><p>Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.</p><p>Conclusion</p><p>Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.</p></div

Genomic DNA release and obtained plasma volume after 3 days of storage within temperature range recommended by the manufacturer (study cohort V).

<p>Blood collected in cfDNA BCTs (n = 8) was stored for 3 days at the indicated temperature and subsequently analyzed using the genomic DNA release assay based on the 402:96 bp LINE-1 ratio (A). Shown are box plots with 1.5 x IQR applied to create whiskers. Statistically significant differences from the reference condition (20°C) were determined by one-way ANOVA and are marked with an * (p ≤ 0.05). (B) Obtained mean plasma volume with SD for indicated storage conditions.</p

Analysis of cfDNA yield and genomic DNA release for study cohort I.

<p>(A) DNA yield was assessed for cfDNA from blood samples stored at room temperature (18°C– 22°C) in K₂EDTA tubes vs cfDNA BCTs (healthy donors, n = 60). Plasma was prepared after indicated storage conditions. Extracted DNA was analyzed for overall yield by qPCR amplifying a 96 bp LINE-1 fragment. (B) Illustration of the DNA yield ratio between long (402 bp) and short (96 bp) LINE-1 fragments (n = 60). Increased ratios compared to K₂EDTA reference would indicate genomic DNA release. Shown are box plots with 1.5 × interquartile range (IQR) applied to create whiskers and outliers. Statistical analysis using one-way ANOVA revealed no significant difference between conditions.</p

Effect of extreme storage temperatures on plasma separation and DNA yield in study cohort IV.

<p>(A) Representative image of cfDNA BCTs centrifuged after 3 days of storage at RT, 4°C and 40°C. RT storage resulted in expected plasma separation with defined buffy coat layer and clear yellow plasma fraction. Extreme temperature conditions resulted in an expanded cellular interface layer or hemolytic plasma at 4°C or 40°C, respectively. (B) Effect of extreme temperatures on genomic DNA release (402 bp LINE-1 qPCR fragment). Statistically significant differences between K₂EDTA and cfDNA BCT storage conditions determined by one-way ANOVA are marked with ** (p ≤ 0.01). Shown are box plots with 1.5 x IQR applied to create whiskers. (C) Obtained mean plasma volume with SD for indicated storage conditions.</p