Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D

High-throughput sequencing has rapidly gained popularity for transcriptome analysis in mammalian cells because of its ability to generate digital and quantitative information on annotated genes and to detect transcripts and mRNA isoforms. Here, we described a double-random priming method for deep sequencing to profile double poly(A)-selected RNA from LNCaP cells before and after androgen stimulation. From Ϸ20 million sequence tags, we uncovered 71% of annotated genes and identified hormone-regulated gene expression events that are highly correlated with quantitative real time PCR measurement. A fraction of the sequence tags were mapped to constitutive and alternative splicing events to detect known and new mRNA isoforms expressed in the cell. Finally, curve fitting was used to estimate the number of tags necessary to reach a ''saturating'' discovery rate among individual applications. This study provides a general guide for analysis of gene expression and alternative splicing by deep sequencing.alternative splicing ͉ androgen-regulated gene expression in prostate cancer cells ͉ curve regression ͉ high-throughput sequencing M icroarray-based approaches, especially unbiased tiling arrays, suggest that up to 80% of the genome may be transcribed to produce a huge number of uncharacterized transcripts relative to current gene annotation (1-4). In contrast, recent transcriptome analysis by deep sequencing indicates that the vast majority of expressed transcripts in mammalian tissues and cell lines are confined to annotated genes and exons (5, 6). Although microarraybased approaches suffer from a great degree of uncertainty in relating detected hybridization signals to defined transcripts, sequencing-based approaches tend to be overwhelmed by abundant transcripts in the cell. Construction of ''normalized'' libraries for deep sequencing might facilitate the discovery of low abundance transcripts, many of which may act as noncoding, regulatory RNA in mammalian cells.An advantage of transcriptome analysis by deep sequencing is the ability to detect structural variation of individual transcripts. It is well known that different transcripts from the same genes may be generated by differential promoter usage, heterogeneous transcriptional start sites and alternative 3Ј end formation (2). A recent Pol II ChIP-chip study indicates that protein-coding genes may have an average of 3 to 5 promoters in both the mouse and human genomes (7). Further adding to the diversity in the transcriptome is alternative RNA processing of most protein-coding genes as a consequence of alternative 5Ј and 3Ј splice site choices, exon inclusion/ skipping, intron retention, and combinatorial use of alternative exons (8). It is estimated that up to 74% of human genes undergo alternative splicing, which is believed to contribute to the complexity of the proteome in mammalian cells (9).It is striking to note that individual laboratories now have the capacity to generate sequenced tags that are on the same order of sequenced mRNA/ESTs in publicly availab...

Section: Resultsmentioning

confidence: 99%

Determination of tag density required for digital transcriptome analysis: Application to an androgen-sensitive prostate cancer model

Lovci

Kwon

et al. 2008

“…The 1,013 aTSS in the Synechocystis 6803 chromosome with 3,172 annotated ORFs (NCBI annotation) demonstrate the level of complexity in the transcriptome of this model organism. To exclude the possibility that the aTSS were experimental artifacts of nonspecific priming during cDNA synthesis (33), the dRNAseq results were compared with available tiling array data (13) and with a transcriptome array in which RNA was labeled directly to avoid such artifacts. This comparison verified the existence of a plethora of antisense transcripts in this organism and recapitulates recent findings of massive antisense transcription in other prokaryotes (34).…”

Section: Discussionmentioning

confidence: 99%

An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803

Mitschke

Georg

Scholz

et al. 2011

350

459

There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5′ annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.gene expression regulation | promoter prediction | RNA polymerase

“…Total RNA from diploid cells was quality controlled with a BioAnalyzer (Agilent) and used to enrich for poly-A + transcripts with the Oligotex kit (Qiagen). Purified polyA + RNA was reverse transcribed in the presence of actinomycin D to prevent spurious antisense RNA synthesis (49). Proprietary high-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) were used for raw data production using the GCS3OOO TG system (Affymetrix) (17).…”

Section: Methodsmentioning

confidence: 99%

Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6

Lardenois

Liu

Walther

et al. 2010

Self Cite

126

217

Budding yeast noncoding RNAs (ncRNAs) are pervasively transcribed during mitosis, and some regulate mitotic protein-coding genes. However, little is known about ncRNA expression during meiotic development. Using high-resolution profiling we identified an extensive meiotic ncRNA expression program interlaced with the protein-coding transcriptome via sense/antisense transcript pairs, bidirectional promoters, and ncRNAs that overlap the regulatory regions of genes. Meiotic unannotated transcripts (MUTs) are mitotic targets of the conserved exosome component Rrp6, which itself is degraded after the onset of meiosis when MUTs and other ncRNAs accumulate in successive waves. Diploid cells lacking Rrp6 fail to initiate premeiotic DNA replication normally and cannot undergo efficient meiotic development. The present study demonstrates a unique function for budding yeast Rrp6 in degrading distinct classes of meiotically induced ncRNAs during vegetative growth and the onset of meiosis and thus points to a critical role of differential ncRNA expression in the execution of a conserved developmental program.