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-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-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-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

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)

New biological outcomes of validated DEGs.

<p>A) <i>FN1</i> is restricted to the endoderm. <i>In situ</i> hybridisation in AI (a), IVP (b), and SCNT D18 EE tissues: Med (c), Low (d), High (e), using an anti-sense FN1 DIG-labelled riboprobe. (t) trophoblast, (e) endoderm. In the AI panel, f) shows c93/<i>SOLD1</i>, a trophoblast-specific control from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038309#pone.0038309-Degrelle1" target="_blank">[23]</a>. The sense probe gave a negative signal in all tissues (not shown). Despite differential expression levels in array and QPCR data, this gene is expressed in the same cells, regardless of conceptus origin. Only a small part of each conceptus is shown. Scale bar is 150 µm. B) Microvilli abnormalities in SCNT EE tissues at D18. In the SCNT groups where <i>MYO6</i> and <i>LHFPL2</i> were underexpressed (b, c), epithelial microvilli appeared shorter and/or fused. SEM images of the external face (extra-embryonic ectoderm or trophoblast) of D18 EE tissues from SCNT Low and Med conceptuses as compared to controls (AI in a). Magnifications: a) x 30000, b) x 35000, c) x 30000. C) <i>PLIN2</i> is expressed in the trophoblast of D18 EE tissues and absent from the yolk sac at D25 [the yolk sac is composed of endoderm (e) and mesoderm (m)]. It is also expressed in binucleated cells (BNC) from D63 bovine placentas. BNC are differentiated trophoblast cells, often considered as the anatomical equivalent of mouse giant cells. <i>In situ</i> hybridisations with an anti-sense PLIN2 DIG-labelled riboprobe on tissue cross-sections from D18 EET (a), D25 Yolk sac (b) and D63 placentas (c) developed after AI. The sense probe gave a negative signal in all tissues; data are not shown. Only a small part of each tissue is shown. Scale bar is 100 µm.</p

Molecular uncoupling of E and EE tissues in SCNT conceptuses.

<p>The gene set used to classify each conceptus includes six genes from the extra-embryonic tissues and has been described as an accurate predictor of embryonic stages in controls. A) Patterns resulting from AI: examples of a filamentous conceptus with a disc at stage 3 (Fil-N2) and a trophoblastic vesicle (TV) with an ablated disc since Day 15. B) Patterns resulting from SCNT: examples of tubular conceptuses with a delayed embryonic disc (stage 2) or no disc (Ab2).</p