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Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.

Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95-100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.

LIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein that is known to be involved in numerous biological and pathological processes. LASP-1 overexpression has been described in several types of cancers, but its expression and role in clear cell renal cell cancer (ccRCC) remains unknown.Using immunohistochemistry, we analyzed LASP-1 protein expression in 216 clinicopathologically characterized ccRCC cases. We also examined LASP-1 expression in 20 paired ccRCC tissues and in 2 cell lines by real-time PCR and Western blot. Using RNA interference, we investigated the effects of LASP-1 depletion on tumor cell behavior in vitro. Statistical analyses were used to determine the associations between LASP-1 levels, tumor features and patient outcomes.LASP-1 overexpression was observed in ccRCC tissues (P

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3' exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3' exons of ZNFs are promoters. We further characterized the histone modifications at the 3' ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3' exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3' ZNF exon was removed from its natural genomic location, the isolated ZNF 3' exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3' exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3' exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3' exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.

Massively parallel sequencing technology now provides the opportunity to sample the transcriptome of a given tissue comprehensively. Transcripts at only a few copies per cell are readily detectable, allowing the discovery of low abundance viral and bacterial transcripts in human tissue samples. Here we describe an approach for mining large sequence data sets for the presence of microbial sequences. Further, we demonstrate the sensitivity of this approach by sequencing human RNA-seq libraries spiked with decreasing amounts of an RNA-virus. At a modest depth of sequencing, viral transcripts can be detected at frequencies less than 1 in 1,000,000. With current sequencing platforms approaching outputs of one billion reads per run, this is a highly sensitive method for detecting putative infectious agents associated with human tissues.

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling. [ABSTRACT FROM AUTHOR]

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3' exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3' exons of ZNFs are promoters. We further characterized the histone modifications at the 3' ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3' exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3' ZNF exon was removed from its natural genomic location, the isolated ZNF 3' exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3' exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3' exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3' exons may function to protect the genome from inappropriate recombination rather than to regulate transcription. [ABSTRACT FROM AUTHOR]