Background: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3-Ribo-SPIA TM primes cDNA synthesis at the 3 polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3 polyA tail. Methods: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip ® array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA TM ). Results: Ribo-SPIA-based global RNA amplification exhibited linearity over 6 orders of magnitude of tran-
Gene expression analysis has become an invaluable tool for understanding gene function and regulation. However, global expression analysis requires large RNA quantities or RNA preamplification. We describe an isothermal messenger RNA (mRNA) amplification method, Ribo-SPIA, which generates micrograms of labeled cDNA from 5 ng of total RNA in 1 day for analysis on arrays or by PCR quantification. Highly reproducible GeneChip array performance (R2 > 0.95) was achieved with independent reactions starting with 5-100 ng Universal Human Reference total RNA. Targets prepared by the Ribo-SPIA procedure (20 ng total RNA input) or the Affymetrix Standard Protocol (10 microg total RNA) perform similarly, as indicated by gene call concordance (86%) and good correlation of differential gene expression determination (R2 = 0.82). Accuracy of transcript representation in cDNA generated by the Ribo-SPIA procedure was also demonstrated by PCR quantification of 33 transcripts, comparing differential expression in amplified and nonamplified cDNA (R2 = 0.97 over a range of nearly 10(6) infold change). Thus Ribo-SPIA amplification of mRNA is rapid, robust, highly accurate and reproducible, and sensitive enough to allow quantification of very low abundance transcripts.
p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.
Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21 BAX is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21 BAX was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21 BAX expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21 BAX induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21 BAX in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21 BAX in glioblastomaderived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.
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