Amplification biases: possible differences among deviating gene expressions.

International audienceBACKGROUND: Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. RESULTS: Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3) and somatic tissues (n = 2), we proceeded to moderate amplifications starting from 1 mug of total RNA (global PCR or IVT one round). Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number) but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70%) and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID). However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated). In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. CONCLUSION: Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i) the sample used: brain, ovary or embryos, (ii) the enzymatic properties initially inferred (exponential or linear) and (iii) the preliminary optimization of the protocols. Moreover the use of an in-house developed array, small-sized but well suited to the tissues we worked with, was of real interest for the search of differential expressions

Amplification biases: possible differences among deviating gene expressions

Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3) and somatic tissues (n = 2), we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round). Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number) but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70%) and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID). However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated). In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i) the sample used: brain, ovary or embryos, (ii) the enzymatic properties initially inferred (exponential or linear) and (iii) the preliminary optimization of the protocols. Moreover the use of an in-house developed array, small-sized but well suited to the tissues we worked with, was of real interest for the search of differential expressions.</p

Amplification biases: possible differences among deviating gene expressions-8

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>nalyse the relevance and biases occurring in our gene profiling studies following moderate RNA amplification: IVT one round or global PCR (details on the protocol in the Methods). In this design technical replicates included both target replicates (independently amplified targets) and array replicates (identical copies of the array)

Amplification biases: possible differences among deviating gene expressions-6

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p> of the core array (or 1 K array; n = 987) whenever interesting. The distributions of EST size, Bt. size and EST positions within the referenced transcripts from the Bos taurus Unigene index (or Bt.) were represented by box plots (A-C). Sizes were expressed in base pairs. Positions within the Bt. were expressed as % of the whole Bt. size, starting from the 5' end which is defined here as 0. The GC content in these subsets and in the corresponding Bt. was also represented by box plots (D-E). Boxes from the box-plots extended from the 25percentile to the 75percentile with a horizontal bar representing the median. Statistical significance between median EST size, Bt. size, EST positions and GC contents has been estimated with T tests (null hypothesis: no differences). (*) means significant (P < 0.05) and (***) highly significant (P < 0.01)

Amplification biases: possible differences among deviating gene expressions-3

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>s and plotted pair-wise per replicate. For example, PCR-amplified: replicate 1 versus replicate 2 (A) and IVT-amplified: replicate 1 versus replicate 2 (B). Similar results were obtained for the 4 other pairs (1 versus 3 and 2 versus 3). Signal mean intensities per protocol were calculated on 12 arrays (4 arrays per replicate × 3 replicates) and plotted pair-wise per protocol (C): PCR-amplified versus IVT-amplified. Similar results were obtained for the ovary and the embryos (ovoid, tubular and filamentous stages). Correlation factors between hybridisation profiles (D)

Amplification biases: possible differences among deviating gene expressions-4

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>per target × 3 targets × 1 tissue = brain) and plotted pair-wise per protocol: PCR-amplified versus IVT-amplified using either the whole set of data (A) or its biological core (B). Coloured, the signals identified as significant gene expression differences between protocols whatever the tissues (n = 154; red: global PCR; blue: IVT one round)

Amplification biases: possible differences among deviating gene expressions-1

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>brane and hybridised with DNA probes encoding exogenous (CG03) or endogenous controls (somatic: EF1α, L23a, cytochrome oxidase III; embryonic: IFN-tau). Each replicate (1 to 3) has been generated independently with each protocol (IVT or PCR) on each tissue (A: brain, ovary; B: ovoid, tubular and filamentous bovine embryos). PolyA+ RNA was used as standard for somatic tissues only (A)

Amplification biases: possible differences among deviating gene expressions-0

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>nalyse the relevance and biases occurring in our gene profiling studies following moderate RNA amplification: IVT one round or global PCR (details on the protocol in the Methods). In this design technical replicates included both target replicates (independently amplified targets) and array replicates (identical copies of the array)

Amplification biases: possible differences among deviating gene expressions-7

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>ages with PCR and IVT amplified samples (A). The distribution of the EST positions within the referenced transcripts from the Unigene Bos taurus index (or Bt.) and the distribution of the EST size were represented by box plots (B, C). Positions within the Bt. were expressed as % of the whole Bt. size, starting from the 5' end which is defined here as 0 (B). Sizes were expressed in base pairs (C). Boxes from the box-plots extended from the 25percentile to the 75percentile with a horizontal bar representing the median. Statistical significance between median EST size or EST positions has not been evaluated

Amplification biases: possible differences among deviating gene expressions-5

<p><b>Copyright information:</b></p><p>Taken from "Amplification biases: possible differences among deviating gene expressions"</p><p>http://www.biomedcentral.com/1471-2164/9/46</p><p>BMC Genomics 2008;9():46-46.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2257942.</p><p></p>erences (n = 154) identified between PCR and IVT amplified samples whatever the tissues (A). Biological processes concerned by these gene expression distortions as defined by a search through Gene Ontologies (B). Representations of the deviating gene expressions on the core array: n = 987 EST as compared to the whole IVT and PCR datasets (C): the black lines correspond to the distribution of the intensities in each dataset; the red lines and the blue lines correspond respectively to densities of the deviating expressions from Panel 1 and 2