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Biologically relevant effects of mRNA amplification on gene expression profiles.

ABSTRACT: BACKGROUND: Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. RESULTS: Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. CONCLUSION: This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

SUBMITTER: van Haaften RI

PROVIDER: S-EPMC1523219 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Biologically relevant effects of mRNA amplification on gene expression profiles.

van Haaften Rachel I M RI Schroen Blanche B Janssen Ben J A BJ van Erk Arie A Debets Jacques J M JJ Smeets Hubert J M HJ Smits Jos F M JF van den Wijngaard Arthur A Pinto Yigal M YM Evelo Chris T A CT

BMC bioinformatics 20060411

<h4>Background</h4>Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material y ...[more]

PMID: 16608515

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Project description:The nycthemeral transcriptome embodies all genes displaying a rhythmic variation of their mRNAs periodically every 24 hours, including but not restricted to circadian genes. In this study, we show that the nycthemeral rhythmicity at the gene expression level is biologically functional and that this functionality is more conserved between orthologous genes than between random genes. We used this conservation of the rhythmic expression to assess the ability of seven methods (ARSER, Lomb Scargle, RAIN, JTK, empirical-JTK, GeneCycle, and meta2d) to detect rhythmic signal in gene expression. We have contrasted them to a naive method, not based on rhythmic parameters. By taking into account the tissue-specificity of rhythmic gene expression and different species comparisons, we show that no method is strongly favored. The results show that these methods designed for rhythm detection, in addition to having quite similar performances, are consistent only among genes with a strong rhythm signal. Rhythmic genes defined with a standard p-value threshold of 0.01 for instance, could include genes whose rhythmicity is biologically irrelevant. Although these results were dependent on the datasets used and the evolutionary distance between the species compared, we call for caution about the results of studies reporting or using large sets of rhythmic genes. Furthermore, given the analysis of the behaviors of the methods on real and randomized data, we recommend using primarily ARS, empJTK, or GeneCycle, which verify expectations of a classical distribution of p-values. Experimental design should also take into account the circumstances under which the methods seem more efficient, such as giving priority to biological replicates over the number of time-points, or to the number of time-points over the quality of the technique (microarray vs RNAseq). GeneCycle, and to a lesser extent empirical-JTK, might be the most robust method when applied to weakly informative datasets. Finally, our analyzes suggest that rhythmic genes are mainly highly expressed genes.

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Biologically relevant effects of mRNA amplification on gene expression profiles.

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Biologically relevant effects of mRNA amplification on gene expression profiles.

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