Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression

1471-2164-7-23 1471-2164 Research article Transcriptome analysis of a cnidarian – dinoflagellate mutualism reveals complex modulation of host gene expression Rodriguez-Lanetty Mauricio rodrigm@science.oregonstate.edu Phillips S Wendy reynoldw@science.oregonstate.edu Weis M Virginia weisv@science.oregonstate.edu

Department of Zoology, Oregon State University, Corvallis, OR 97331, USA

Centre for Marine Studies, University of Queensland, St. Lucia, QLD 4072, Australia

BMC Genomics 1471-2164 2006 7 1 23 http://www.biomedcentral.com/1471-2164/7/23 16472376 10.1186/1471-2164-7-23 16 9 2005 10 2 2006 10 2 2006 2006 Rodriguez-Lanetty et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Cnidarian – dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian – algal symbiosis.

Results

We detected statistically significant differences in host gene expression profiles between sea anemones (Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian – dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress.

Conclusion

Our data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian – dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

Background

Mutualistic symbioses are defined as the association between unrelated organisms living together in a close, protracted relationship that benefits both partners. They are common in nature and are a driving force in evolution 1. Cnidarian – dinoflagellate associations represent one of the most important symbioses in the marine environment. These partnerships form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species.

The cnidarian host harbors algal endosymbionts, usually from the genus Symbiodinium, within gastrodermal cells in vacuoles of phagosomal origin known as the symbiosome. The initial infection occurs when host gastrodermal cells lining the gastric cavity phagocitize algal symbionts previously ingested through the host mouth during feeding 2. The mechanisms of avoiding host digestion remain largely unknown, although some studies suggest that persistence may be due to the failure of phagosome-lysosome fusion 3456.

This intracellular association is centered around nutrient exchange and is essential for both partners to thrive in tropical seas, an environment particularly low in nutrients. The algal endosymbionts can translocate up to 95% of their photosynthetic products to the hosts, where these compounds are primarily used to meet host respiratory demand 78. In return, the symbionts receive protection from predation, nitrogen-based nutrients released from the host 8, and inorganic carbon for photosynthesis 910.

Despite the prevalence of these marine symbioses and the overall interest in coral reef health, we still know very little about the cellular and molecular basis of the intracellular cnidarian – dinoflagellate symbiosis. What are the key molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities? To date, only a few studies have examined either broad-scale patterns of RNA or protein expression in symbiotic cnidarians or identified specific genes that play a role in interpartner communication and regulation. Two dimensional proteomic analysis comparing symbiotic and naturally occurring symbiont-free (aposymbiotic) individuals of the Pacific coast temperate anemone Anthopleura elegantissima revealed dozens of proteins that were up- or down-regulated in the symbiotic state 11. In subsequent studies, one strongly expressed symbiotic protein, sym32, was identified as belonging to the Fasciclin I protein family some of which function in cell-cell interactions or cell adhesion in other organisms 12. In addition, immunocytochemistry and immunoblot studies using an anti-sym32 antibody found a putative homolog in the symbionts, leading to the suggestion that host-symbiont heterophilic fasciclin I interactions could be a method of interpartner signaling in the symbiosis 13. Finally, in a recent study, a lectin has been identified and characterized from a symbiotic soft coral that may play a role in lectin-glycan signaling during onset of symbiosis 14.

The discovery and identification of host genes that modulate cnidarian – dinoflagellate symbioses is a topic that is ideally suited to a comprehensive microarray approach. Recently, similar approaches have been used successfully to identify "symbiosis-specific" genes in other mutualistic associations such as rhizobial 15 and arbuscular mycorrhizal 16 symbioses. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian – algal symbiosis. Following earlier proteomic studies, we used the temperate anemone Anthopleura elegantissima as a model as it occurs naturally in both the symbiotic and aposymbiotic state (Figure 1). This first comprehensive molecular examination of the cnidarian – dinoflagellate association provides critical insights into the maintenance and regulation of the symbiosis.

Figure 1

Photograph of a symbiotic (left) and aposymbiotic (right) Anthopleura elegantissima

Photograph of a symbiotic (left) and aposymbiotic (right) Anthopleura elegantissima.

Results and discussion

A total of 583 (5.62%) of the 10,368 features from the cDNA microarray were identified as significantly different between aposymbiotic and symbiotic anemones (P < 0.01). However, since 104 false positives were expected from the 10,368 gene-tests performed under the null hypothesis when P = 0.01, we applied a False Discovery Rate (FDR) multiple-testing adjustment to control for type I error 17. Allowing for a 5% type I error for the whole set of significant genes, only 189 features, or 1.82% of the original 10,368 features, showed significant differences in expression between the aposymbiotic and symbiotic anemones (P < 0.05; Figure 2). After DNA sequencing and sequence analyses, these 189 features resolved into 91 unigenes.

Figure 2

Volcano plot displaying the probability and ratio of gene expression of all the 10,368 features on the array

Volcano plot displaying the probability and ratio of gene expression of all the 10,368 features on the array. The spots above the probability threshold p < 0.05 (horizontal solid line) represent the 189 significantly different features. For better visualization, the log probability axis is shown with positive numbers

Twelve of the 91 differential unigenes were very highly expressed in symbiotic state (Figure 3). These were viewed with suspicion as possible algal genes that were contaminating the host-only cDNA library. Specific primers for these highly symbiotic unigenes were constructed and used in PCR reactions with host-only and algae-only genomic DNA. Successful DNA amplification was achieved only in the algal DNA samples and not in the host genomic DNA (data not shown). These contaminating algal unigenes were therefore removed from further analyses.

Figure 3

Distribution of unigenes as a function of the ratio of expression between symbiotic and aposymbiotic anemones

Distribution of unigenes as a function of the ratio of expression between symbiotic and aposymbiotic anemones. Arrows divide those down- and up-regulated in symbiosis. Black bars denote unigenes confirmed by PCR to be contaminating algal unigenes (see text for details).

The remaining 79 host unigenes showed differences in expression mostly ranging between 1 and 2 fold change. We hypothesize that these subtle changes in expression could be due in part to the use of whole animal mRNA extractions for the screening of the arrays. As the symbionts reside solely in the gastrodermal tissue of the host, a larger fold change of expression in the tissues which harbor symbionts could be hidden by non-differential expression of the same genes in non-symbiotic tissue. However, if genes were turned on or off in the whole animal as a function of symbiosis, we still would expect to see large changes in expression from the array data. We did not see such large fold changes of any genes. Future experiments comparing expression only in the gastrodermal tissue from symbiotic and non-symbiotic animals might show greater differences in expression in the same genes we describe here.

From our 79 identified host unigenes, only 28 (35%) showed significant BLAST hits (E < 1.0 × 10^-4) with homologs to known genes in the Genbank (Table 1). Twenty three of these 28 genes matched more strongly to vertebrate homologs than to invertebrate homologs. This result is consistent with previous findings suggesting that cnidarian genomes contain many genes previously considered to be vertebrate innovations because of their absence from the Drosophila or Caenorhabditis genomes 18. This provides further evidence for the unexpected paradox of genome evolution pointed out by Kusserow et al. 19: "the gene diversity in the genomes of simple metazoans is much higher than previously predicted and some derived lineages such as flies and nematodes have a lower gene family diversity than simple metazoans."

Table 1

Known unigenes showing significant expression differences between symbiotic and aposymbiotic anemones. Identification was based on significant blast hits (E values < 10^-4) from GenBank. Ratios of expression from all microarray analyses and selected Q-RT-PCR analyses are shown. Arrows indicate the direction of expression as a function of symbiosis: up- or down-regulation. Those genes highlighted in bold are discussed in the text. Significance is indicated as follows: P < 0.05 (*), P < 0.01 (**).

Unigenes grouped based on functional category

Genbank Accession ID

OSU-CGRB ID

Expression as function of symbiosis

Microarray

Q-RT-PCR

Cell Growth and Maintenance

von Willebrand factor

DQ309533

AeOSU006130

↑ 1.92 *

↑ 1.82 *

Tubulin alpha chain

DQ309523

AeOSU007825

↓ 1.20 *

Myosin regulatory light chain 2

DQ309534

AeOSU008456

↓ 1.37 *

Beta-tubulin

DQ309552

AeOSU009795

↓ 1.39 *

Collagen alpha-1 chain, type XII

DQ309546

AeOSU000073

↓ 1.94 **

Metabolism and Energy Pathways

Phytanoyl-CoA hydroxylase

DQ309528

AeOSU005830

↑ 1.54 *

↑1.59 *

Selenoprotein W1

DQ309535

AeOSU002895

↑ 1.42 *

Long chain acyl-CoA thioesterase

DQ309538–DQ309539

AeOSU003383

↑ 1.28 *

ATP synthase, H⁺transporting, mitochondrial F0 complex

DQ309536

AeOSU000538

↓ 1.29 *

Medium chain S-acyl fatty acid synthetase thio ester hydrolase

DQ309537

AeOSU000658

↓ 1.56 *

↓ 1.32 *

Copper/zinc superoxide dismutase (SOD)

DQ309550

AeOSU001001

↓ 1.58 *

↓ 1.89 *

Protein metabolism

60S ribosomal protein L26

DQ309540

AeOSU004867

↑ 3.43 **

Plasma glutamate carboxypeptidase

DQ309521

AeOSU001186

↑ 1.70 *

Nucleic Acid Metabolism

Activating transcription factor 4 (ATF4)

DQ309526

AeOSU001247

↑ 1.51 *

↑ 2.49 *

Histone H3.3

DQ309522

AeOSU007640

↑ 1.22 *

DEAD box polypeptide 48

DQ309551

AeOSU006260

↓ 1.30 *

Signal Transduction &cell communication

Oncoprotein-induced transcript 3

DQ309527

AeOSU009888

↑ 1.40 *

Scavenger receptor class B, type 1 (CD36)

DQ309525

AeOSU008133

↑ 1.34 *

Sphingosine 1-phosphate phosphatase 2

DQ309531–DQ309532

AeOSU005519

↓ 1.50 *

↓ 1.63 *

Betaine homocysteine methyl transferase

DQ309529

AeOSU002910

↓ 1.56 *

↓ 4.18 *

Calcium/calmodulin-dependent protein kinase IV

DQ309544

AeOSU005891

↓ 1.57 *

Prohibitin protein

DQ309542–DQ309543

AeOSU001676

↓ 2.21 *

Transport

Membrane-bound transferrin-like protein p97

DQ309541

AeOSU001619

↓ 1.29 *

Unclassified

Death-associated protein 1 (DAP-1)

DQ309530

AeOSU008955

↑ 1.39 *

Human headcase homolog (HECA)

DQ309548

AeOSU009119

↑ 1.33 *

Guanine nucleotide binding protein, gamma 5 or 12

DQ309547

AeOSU008880

↑ 1.29 *

SEC31-like 2 isoform A

DQ309524

AeOSU009211

↑ 1.17 *

Methylmalonate semialdehyde dehydrogenase

DQ309545

AeOSU005938

↓ 1.37 *

Fifty one (65%) of our identified unigenes are unknown. This is consistent with other EST projects of other cnidarians that show a high number of unknown genes; 30% in the scleractinian coral Acropora millepora 18 and ~44% in the anemone Aiptasia pulchella 20

Rather than finding "symbiosis' genes which change expression as a function of symbiosis in an ON/OFF manner, we detected instead alterations in expression of genes regulating a broad array of functional processes (Figure 4, Table 1). Functional gene classification of the 28 differentially expressed known genes reveals the complex effect of symbiotic state on host gene expression. This suggests that symbiosis is regulated and controlled by changes within existing pathways used to control metabolism and growth of the animal as a whole rather than by pathways unique to the symbiotic state. Interestingly, despite the differences among mutualistic symbioses in nature, the molecular modulation of existing pathways in the host cell seems to be a key common factor in symbiosis regulation 212223. The functional interpretation of the 28 differentially expressed genes based on their functional classification led us to examine the pivotal expression changes involved in: 1) lipid metabolism, 2) cell proliferation and apoptosis, and 3) oxidative stress in the cnidarian – Symbiodinium symbiosis.

Figure 4

Distribution of the known host unigenes based on their functional classification

Distribution of the known host unigenes based on their functional classification. White and grey bars are the genes up- and down-regulated in symbiosis, respectively.

Alterations in lipid metabolism

The mutualistic interaction between cnidarians and Symbiodinium has a nutritional basis involving the sustained and substantial bidirectional translocation of nutrients between the algal and animal host cells. It is well known that algae can release much of their photosynthetic carbon (>90%) to the animal host 7. These photosynthates include carbohydrates, in the form of glucose 24, glycerol 25 and saturated and poly-unsaturated fatty acids 2627. In this study, expression changes of enzymes involved in host lipid metabolism provide indirect evidence suggesting that symbiotic anemones are indeed processing more lipids than are aposymbiotic anemones.

Phytanoyl-CoA hydroxylase, an enzyme involved in lipid degradation, which catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA in beta-methylated fatty acid metabolism 28, was more highly expressed in symbiotic compared to aposymbiotic anemones (1.54 fold from array data and 1.59 fold from Q-RT-PCR data). In contrast, the enzyme medium-chain S-acyl fatty acid synthase, involved in lipid synthesis, was down-regulated in symbiosis (1.56^-1fold from array data and 1.32^-1fold from Q-RT-PCR). Similar up-regulation of degradative enzymes and down-regulation of lipid synthesis enzymes, particularly the fatty acid oxidizing enzymes, have been detected in mammals in response to exogenous supply of fatty acids 29. In the context of cnidarian – dinoflagellates symbioses, it makes sense that the host would not need to synthesize certain lipids if they are supplied from the symbiont, and that the host would turn on degradative machinery to break down the supplied lipids. In addition, we detected an up-regulation of the long chain acyl-CoA thioester hydrolase enzyme (1.28 fold from array data), the activity of which is also normally increased in mammalian animals when diets rich in polyunsaturated fatty acids are supplied 30. This enzyme has also been implicated as an important signaling molecule in the regulatory cascade leading to fatty acid-mediated alterations in gene expression 29. Further functional studies on cnidarian fatty acyl-CoA thioesters could shed light on the role of this compound in regulation of symbiosis.

The scavenger receptor class B type I (SR-BI, a member of the CD36 superfamily) is another differentially expressed gene also implicated in lipid metabolism. We detected an up-regulation of this scavenger receptor in symbiotic state (1.34 fold array data). SR-BIs are integral membrane glycoproteins found on the surface of a variety of cells that have functions ranging from fatty acid translocation to cell adhesion. We will discuss how the different functions described in other biological systems might also play a role in cnidarian – algal symbioses.

SR-BIs function in selective uptake and transport of cholesterol and other fatty acids from lipoproteins 31. In the context of cnidarian – dinoflagellate symbioses, a cnidarian SR-BI homolog could facilitate traffic of the symbiont-derived fatty acid within and between host cells. Such a role is consistent with the increased expression of this protein in the symbiotic state, and with evidence that lipids are translocated from the algal symbiont to the host cell 27.

SR-BI (CD36) proteins are also involved in pathogen/parasite infection and cell adhesion. Recent evidence suggest that some pathogens such as Mycobacterium 32 and Hepatitis C virus (HCV) 3334 take advantage of host SR-BI proteins and use them during the invasion of host cells. More specifically, Voisset et al. 34 showed a role in lipid transfer during HCV entry, which involves a complex interplay between SR-BI, high density lipoprotein and HCV envelop glycoproteins. Further, host SR-BIs facilitate adhesion of Plasmodium-infected human erythrocytes to uninfected erythrocytes, allowing for movement of the parasite between host cells 35. A cnidarian SR-BI homologs could perform a similar function in facilitating dinoflagellate symbiont infection and transport from infected to uninfected host cells. This function is particularly intriguing with regard to cnidarian – dinoflagellates symbioses as Plasmodium is a member of the apicomplexa, a sister taxon to the dinoflagellates 3637. Future studies of immuno-fluorescence microscopy and immuno-inhibition of the cnidarian SR-BI homolog would help to localize and describe the function of this protein in cnidarian – algal symbiosis.

Cell cycle regulation and suppression of apoptosis

The difference in expression between symbiotic and aposymbiotic animals of a key sphingolipid regulator suggests that symbiotic state contributes to suppression of apoptosis and expedition of host cell cycle progression. Transcripts of the enzyme sphingosine-1-phosphate phosphatase (SPPase), which is involved in the regulation of the sphingosine – sphingosine-1-phosphate (S1P) rheostat, were significantly down-regulated in symbiotic anemones (1.5^-1fold from array data and 1.62^-1fold from Q-RT-PCR data). The sphingosine – S1P rheostat determines whether a cell survives and proliferates or undergoes apoptosis and dies. It is regulated by two enzymes, sphingosine kinase which converts sphingosine to S1P and SPPase which catalyzes the opposite reaction.

The sphingosine – S1P rheostat plays multiple signaling roles in higher animals (Figure 5a) [reviewed in 38]. Increased levels of anti-apoptotic S1P enhance cell proliferation by expediting the G1/S transition in the cell cycle and by increasing DNA synthesis through the activation of transcription factors via extracellular-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways 39. S1P also promotes the activation of the transcription factor nuclear factor κB (NF-κB), which plays a role in preventing apoptosis and enhancing cell survival 40. Further, increased levels of S1P suppress apoptosis induced by high levels of pro-apoptotic sphingosine 3839

Figure 5

Cellular regulation of Sphingosine-1-Phosphate (S1P) and Prohibitin

Cellular regulation of Sphingosine-1-Phosphate (S1P) and Prohibitin. A. Sphingosine (Sph) – S1P rheostat; showing the two enzymes that control it, sphingosine kinase and S1P phosphatase (SPPase); and regulation pathways of S1P and prohibitin in vertebrate cells. B. Model of S1P and prohibitin roles in cnidarian – algal symbiosis: (1) SPPase is down regulated by an unknown algal-induced mechanism, causing an increase of the levels of S1P over sphingosine; (2) the lower levels of sphingosine produce a suppression of sphingolipid-induced apoptosis; (3) higher levels of S1P enhance the activation of NF-κB, which inhibits apoptosis and increases cell survival; (4) S1P activates the ERK/MAPK pathway, which causes an expedition of G1/S cell cycle that results in cell proliferation; (5) Prohibitin is down-regulated by an unknown algal-induced mechanism, causing a suppression of apoptosis and a release of the blockage of E2F transcription factors. The latter enhances the expedition of G1/S cell cycle resulting in cell proliferation.

Based on the functions of the sphingosine – S1P rheostat in other organisms we have developed a model for its role in cnidarian – dinoflagellate symbioses (Figure 5b). We hypothesize that, by an unknown mechanism, the symbiotic state causes a down-regulation of host cell SPPase, thereby favoring the accumulation of S1P and reducing levels of sphingosine. High levels of S1P would in turn start a signaling cascade resulting in survival and proliferation of the host cell containing algae. Correspondingly low levels of sphingosine would result in a failure to initiate the apoptosis cascade, thereby promoting host cell survival.

Our model proposes regulation of the sphingosine – S1P rheostat by controlling levels of SPPase. If true, this would be only the second piece of evidence suggesting that the rheostat can be controlled via SPPase levels. Just one other study has demonstrated accumulation of S1P in mammalian cells by gene knockdown of SPPase 41. All other studies of rheostat control have focused on changes in sphingosine kinase activity 42. However, it is possible that the sphingosine kinase transcripts were not present in our anonymous cDNA array, and because of this we can not discard the possibility that this enzyme might also play a role in the modulation of the rheostat as a function of symbiosis.

The hypothesis that host/symbiont interactions are controlled in part by manipulation of host cell survival has been proposed in many pathogenic and parasitic interactions in animals [reviewed in 43] and plants 44. Of particular interest to our study is evidence that the tomato pathogen Pseudomonas syringae up-regulates the expression of a host cell signaling sphingolipid 45. This in turn suppresses programmed cell death of the infected host cell thereby blocking an antimicrobial defense strategy used by many plant hosts.

In addition to observing symbiosis-specific differences in SPPase levels, we also observed a 2.2-fold down-regulation in the symbiotic state of prohibitin, another gene involved in the regulation of the cell cycle and apoptotic pathways (Figure 5a). Prohibitin is a highly conserved protein that inhibits cell proliferation and is a potential tumor repressor 4647. It is thought to act by repressing the E2F family of transcription factors, which promote the G1/S transition 48. Furthermore, increases in prohibitin levels have been correlated with the initial events of apoptosis, however its molecular mechanism of action remains unclear 49. Decreased expression of prohibitin in the symbiotic state fits well into our model for control of host cell proliferation (Figure 5b). Decreased prohibitin would result in increased transition into S phase and inhibition of apoptosis, resulting in symbiotic host cell survival and proliferation.

Oxidative stress

We detected a down-regulation of a copper/zinc superoxide dismutase (CuZnSOD) in the symbiotic state (1.58^-1fold from array data and 1.89^-1from Q-RT-PCR data). Superoxide dismutases function as antioxidants that break down harmful superoxide radicals. In symbiotic cnidarians, a sequence for CuZnSOD has been characterized 50, and two other classes, MnSOD and FeSOD, have been described electrophoretically 51. Previous findings have measured SOD enzyme activities nearly two orders of magnitude greater in symbiotic compared to aposymbiotic Anthopleura elegantissima 52. One possible explanation for this apparently contradictory result is that the other SOD classes are being expressed specifically in the symbiotic state to mitigate the hyperoxic environment normally encountered in symbiosis. This explanation is in agreement with findings that show a low activity of CuZnSOD in the symbiotic anemone Anemonia viridis but an increased activity of FeSOD and MnSOD isoforms 51. However, it has not been fully demonstrated that these Fe/Mn SODs are indeed of host origin. An alternative explanation for our results is that the host in the symbiotic state gains antioxidant protection from its symbionts, causing a reduction in expression of its own SODs. This would explain the down-regulation of CuZn SOD in the symbiotic state. Future studies may explore the origin of other symbiosis-induced SOD classes.

Conclusion

In summary, we demonstrate that the gene expression profile of the host sea anemone Anthopleura elegantissima changes in symbiosis with the dinoflagellate Symbiodinium muscatinei. The group of genes whose expression is altered is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. Our data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. On the contrary, it appears that the maintenance of the symbiosis is modulated by the alteration of expression of existing genes involved in vital cellular processes.

We show evidence that the gene expression of key biomolecules involved in cell cycle progression and apoptosis are differentially modulated in symbiosis. These results lead us to hypothesize that a suppression of apoptosis together with a deregulation of the host cell cycle create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. These findings have changed our perception of the cellular interaction between cnidarians and symbiotic dinoflagellates. We have always defined the cnidarian – algal interaction as cooperative since the ecological outcome of the interaction is a mutualistic symbiosis. However, at the cellular level the interaction between host and symbiont appears to have components of a parasitic or pathogenic interaction. Mutualistic symbionts, like pathogens, must overcome the host innate immunity to enter, reside, and grow inside the host cell. But intriguingly, though the algae-induced changes in the host cell show some similarity to pathogen – host interactions, they do not lead to the development of disease. This may be a common phenomenon in symbiosis, as a comparable picture is emerging from insect – bacteria 22 and squid – bacteria 53 symbioses. Understanding the nature of the molecular regulation of cnidarian – algal symbiosis by comparison with host/pathogen and host/parasite associations, it will provide further insight into the evolution of symbioses.

Methods

Collection and maintenance of experimental organisms

Specimens of Anthopleura elegantissima (Brandt) were collected in Neptune Beach, Oregon. Naturally-occurring aposymbiotic animals were collected from the intertidal zone in the shaded overhangs underneath the boulders. Symbiotic animals were collected from adjacent sun-exposed rocks. To standardize the physical condition to which the anemones were acclimated, the animals were maintained in the laboratory for four weeks in tanks of running seawater at ambient temperature (~14°C) and a light intensity of 100 μmol quanta m^-2s^-1on a 12 h light/ 12 h dark cycle. Animals were fed brine shrimp once a week. After this 4-week period, animals were frozen in liquid N₂and stored at -80°C. Algal dinoflagellates harbored by the symbiotic anemones were genetically identified, as Symbiodinium muscatinei sensu LaJeunesse and Trench 54 by sequencing and analyzing algal ITS rDNA following Rodriguez-Lanetty and Hoegh-Guldberg's protocol 55.

Construction of cDNA arrays

Host-only RNA from four field collected aposymbiotic (apo) and symbiotic (sym) anemones was used to create apo and sym cDNA libraries in the Lamda Zap II bacteriophage vector (Stratagene). Algal symbionts were extracted and removed from sym anemones before host RNA extraction using previously described methods 12. The Bluescript phagemid was excised and the plasmid library was plated onto LB-agar plates. Individual colonies were picked and grown in LB overnight in 384 well plates. The cDNA inserts were PCR amplifed using M13 forward and reverse vector primers. The size of cDNA inserts varied between 0.4 and 2.5 Kb. The redundancy of the libraries was checked by sequencing random clones. Within 73 sym clones, the redundancy was 16.4%; within 80 apo clones, the redundancy was 18.8%; and within all 153 clones, the redundancy was 29.4%.

To avoid bias in the random sampling of cDNA genes from Anthopleura elegantissima, similar proportions of library clones from the two conditions to be compared (aposymbiotic and symbiotic) were arrayed. A total of 10,368 PCR amplified cDNAs buffered in 3× SSC and 1.5 M betaine were spotted on UltraGAPS™ coated slides (Corning) without duplication. After printing, the arrays were dried for 48 h in vacuum desiccators, and UV cross linked at 300 mJ. The slides were stored in desiccators until the hybridization process.

Hybridization of arrays

For probe construction, total RNA was extracted from aposymbiotic and symbiotic anemones (which included both host and algal partners) using Trizol, and then mRNA was isolated using the MicroPoly(A) Pure kit (Ambion). To confirm that samples were either symbiotic or aposymbiotic and to test sample quality, an initial cDNA synthesis was performed from the total RNA. That cDNA was used in quantitative RT-PCR reactions with anemone-specific actin primers and Symbiodinium rRNA primers. Symbiotic samples were used only if both actin and Symbiodinium rRNA amplified in high levels. In contrast, aposymbiotic samples were used only if actin was amplified in high levels and no amplication was detected of Symbiodinium rRNA.

cDNA probe synthesis was performed from 1 ug mRNA using Powerscript Reverse Transcripase (Clontech) and the Genisphere 3DNA-50 microarray kit according to the manufacturers' instructions. Slides for hybridization were chosen randomly from the batch of high quality printed arrays. Before hybridization, the slides were placed in isopropanol for 15 min, transferred to boiling water for 5 min, and dried via centrifugation. Apo and sym cDNAs were combined and hybridized to arrays in a formamide-based hybridization buffer under LifterSlips overnight at 50°C. Following post-hybridization washes, Cy3 and Cy5 Capture Reagents were hybridized to the array in the formamide-based buffer under LifterSlips for 3 h at 50°C. Following post-hybridization washes, slides were scanned using a GenePix ^®4200 scanner (Axon Instruments) and image acquisition and quality control was performed using the software GenePix ^®Pro 5.

Experimental design and statistical analysis of microarray data

We applied a multiple dye-swap experimental design for the two conditions, aposymbiotic and symbiotic anemone groups, compared in our experiment 56. Six biological replicates per condition were used as recommended for this type of two-comparison experimental design 17 (a total of 11 microarrays, [Gene Expression Omnibus: GSE3958]). Ratio-Intensity plots were constructed for each array data to explore whether or not intensity dependence of log ratios, which appears as curvature, was present. The assumption from cDNA microarray data is that most genes are not differentially expressed among treatments, and therefore most points in the RI plots should fall along a horizontal line centered on zero. Because curvatures were detected in a few of the arrays, an rLowess curve fitting transformation 57 was applied to the data. The transformation was applied to all the arrays to keep consistence in the whole data set as suggested by Cui et al. 58. To detect differentially expressed genes between the two conditions, the following 2-stage ANOVA mixed model 17 was fitted to the log transformed intensity data; Y= A (Array) + D (Dye) + S (Sample = Individual) + C (Condition) + E (Error). In this mixed model, the arrays and samples were treated as random factors, and dye and condition as fixed factors. The Fs statistic, a shrinkage estimator for gene-specific variance components that makes no assumptions about the distribution of variances across genes, was estimated 59. After the statistics were estimated, p-values were calculated from random permutations (N. permutations = 500 with sample reshuffling), and adjusted to correct for Type I error derived from multiple testing using the false discovery rate (FDR) approach 17. All the analyses described above were conducted using the software R/Maanova 17.

Sequencing and gene identity

Differentially expressed cDNA inserts were sequenced from both directions. Using the Seqmerge program from the Wisconsin Package Version 10.3 (Accelrys Inc., San Diego, CA), vector and low quality sequences were masked manually and sequence fragments were assembled into contigs. The contigs and their fragments were manually checked within Seqmerge to ensure an accurate consensus sequence. A blastx search of NCBI non-redundant proteins was performed for each contig, and those with expect values of less than 1 × 10^-4were considered to be homologs to the matched known protein. For each gene, we report the hit with the lowest expect values that are to a known protein.

Validation by quantitative real time PCR

Specific primers amplifying approximately 100–200 bp PCR products were designed for some of the genes as indicated later in the text, and specific amplification of appropriately sized bands was checked (Table 2). cDNAs from each of five aposymbiotic and symbiotic anemones were synthesized from 100 ng of mRNA and then diluted to a final volume of 330 μl. Two μl of cDNA was used in triplicate 20 μl quantitative RT-PCR reactions with 250 nM primers and iQ SYBR Green Supermix (BioRad) for a total of 40 cycles. The comparative delta CT method corrected for the actual PCR efficiency was used to determine relative quantities of mRNA transcripts from each sampled anemone. The PCR efficiency was determined using LinRegPCR 60. For normalization purposes, we used multiple house-keeping genes and calculated a normalization factor from the geometric mean of their expression levels, as proposed by Vandesompele et al. 61. To identify house-keeping gene controls, we selected 8 genes from our microarray platform that did not show significant differences between the treatments and whose ratios of expression were not significantly different from one (single t-test, α = 0.05). These putative house-keeping genes were then tested for their expression stability using geNorm 61. The three most stable genes were used to calculate the normalization factor for each of the cDNA samples. These three house keeping genes are beta-actin [GenBank: DQ314617], a putative senescence-associated protein [GenBank: DQ314618], and ribosomal protein L12 mRNA [GenBank: DQ314616] (Table 2). Statistical analysis was conducted using a permutation t-test on the normalized data.

Table 2

Forward and reverse primers used to amplify the following genes in Q-RT-PCR assays. House keeping genes are indicated with (*).

Gene

Primer sequence

von Willebrand factor

For: CTG CCA AGA GGC GAA TAT GT Rev: GCA CAT GTT CTG GTC CCT CT

Phytanoyl-CoA hydroxylase

For: GCC AGC GCA CAC TGT TAT TA Rev: TTT CCT TGC ACA AGA CGT TG

Medium chain S-acyl fatty acid synthetase thio ester hydrolase

For: GAT AAA ACA CTG GGG AGG AAC Rev: CTT GTT ACT TGA CTC CAA CCT TC

Copper/zinc superoxide dismutase (SOD)

For: CTC AGG AGG GTG ATG GAA AA Rev: TTG CCG AAG GGA TTA AAG TG

Activating transcription factor 4 (ATF4)

For: GCG TCC AAG AGA GAA ACA CC Rev: CGA TAT CTT GTG GCA GCA TC

Sphingosine 1-phosphate phosphatase 2

For: TTG TGG CAC GCA CTC TTT AC Rev: TCC CAT TGG TAT CGC TTT TC

Betaine homocysteine methyl transferase

For: ACC AGG TGG GAT ATG CAA AA Rev: CCC CAC ATT TCA TGC TTT TC

Beta-actin *

For: CTG ATG GAC AGG TCA TCA CCA T Rev: CTC GTG GAT ACC AGC AGA TTC C

Putative senescence-associated protein *

For: TCG CAC CAT TGG CTT TTC Rev: CCT ATC ATT GCG AAG CAG AA

Ribosomal protein L12 mRNA *

For: CCT AAG TTC GAC CCC AAT GA Rev: TTT TGG ATG GTC AGT TGG AC

Authors' contributions

MRL conceived the experimental design and carried out part of the molecular genetic study, performed the statistical analyses, and drafted the manuscript. WSP carried out most of the molecular genetic study, performed the sequence alignments and gene database blasts. VMW conceived the study, and participated in the design and coordination of the study. All the authors read and approved the final manuscript.

Acknowledgements

We would like to thank Caprice Rosato at the Center for Genome Research and Biocomputing in Oregon State University for her invaluable help printing the cDNA microarrays; and Monica Medina and Jodi Schwarz at the Joint Genomic Institute for helping with some of the DNA sequencing for this study. We are especially grateful to Jason Podrabsky at Portland State University for introducing us into the cDNA microarray world. We would also like to thank to the Bayne's lab for allow us to use their array scanner, and the Weis lab members Simon Dunn, Santiago Perez, Elisha Wood-Charlson, Laura Hauck and Joanne Odden for meaningful discussions and comments on early versions of this manuscript. This research was funded by the National Science Foundation (IBN: 0342585) to V. M. W.

Margulis L Symbiosis in Cell Evolution Freeman, New York 2 1993 Cellular events in the reestablishment of a symbiosis between a marine dinoflagellate and a coelenterate Colley NJ Trench RK Cell Tissue Res 1985 239 93 103 10.1007/BF00214908 Endocytosis of the symbiotic dinoflagellate <it>Symbiodinium microadriaticum </it>Freudenthal by endodermal cells of the scyphistomae of <it>Cassiopeia xamachana </it>and resistance of the algae to host digestion Fitt WK Trench RK J Cell Sci 1983 64 195 212 6141174 Molecular identification of Rab7 (ApRab7) in <it>Aiptasia pulchella </it>and its exclusion from phagosomes harboring zooxanthellae Chen MC Cheng YM Sung PJ Kuo CE Fang LS Biochem Biophys Res Commun 2003 308 586 595 10.1016/S0006-291X(03)01428-1 12914791 Molecular cloning of Rab5 (ApRab5) in <it>Aiptasia pulchella </it>and its retention in phagosomes harboring live zooxanthellae Chen MC Cheng YM Hong MC Fang LS Biochem Biophys Res Commun 2004 324 1024 1033 10.1016/j.bbrc.2004.09.151 15485657 ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the <it>Aiptasia – Symbiodinium </it>endosymbiosis Chen MC Hong MC Huang YS Liu MC Cheng YM Fang LS Biochem Biophys Res Commun 2005 338 1607 1616 10.1016/j.bbrc.2005.10.133 16288726 Reef coral energetics: primary production and photoadaptation Porter J Muscatine L Dubinsky Z Falkowski PG Proc R Soc Lond B 1984 222 161 180 The Role of Sym-biotic Algae in Carbon and Energy Flux in Reef Corals Muscatine L Coral Reefs Ecosystems of the World Elsevier Dubinsky Z 1990 25 75 87 A "CO2 supply" mechanism in zooxanthellate cnidarians: role of carbonic anhydrase Weis VM Smith GJ Muscatine L Mar Biol 1989 100 195 202 10.1007/BF00391958 Effect of dissolved inorganic carbon concentration on the photosynthesis of the symbiotic sea anemone <it>Aiptasia pulchella </it>Carlgren: role of carbonic anhydrase Weis VM J Exp Mar Biol Ecol 1993 174 209 225 10.1016/0022-0981(93)90018-J Differential protein profiles reflect the different lifestyles of symbiotic and aposymbiotic <it>Anthopleura elegantissima</it>, a sea anemone from temperate waters Weis VM Levine RP Journal of Experimental Biology 1996 199 883 892 9318671 Symbiosis-enhanced gene expression in cnidarian-algal associations: cloning and characterization of a cDNA, sym32, encoding a possible cell adhesion protein Reynolds WS Schwarz JA Weis VM Comp Biochem Physiol Mol Integrat Physiol 2000 126 33 44 10.1016/S0742-8413(00)00099-2 Localization of a symbiosis-related protein, sym32, in the <it>Anthopleura elegantissima-Symbiodinium muscatinei </it>association Schwarz JA Weis VM Biol Bul 2003 205 339 350 Octocoral chemical signaling selects and controls dinoflagellate symbionts Koike K Jimbo M Sakai R Kaeriyama M Muramoto K Ogata T Maruyama T Kamiya H Biol Bul 2004 207 80 86 Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays Colebatch G Kloska S Trevaskis B Freund S Altmann T Udvardi MK Mol Plant-Microbe Interactions 2002 15 411 420 Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of an arbuscular mycorrhizal symbiosis Liu JY Blaylock LA Endre G Cho J Town CD VandenBosch KA Harrison MJ Plant Cell 2003 15 2106 2123 181334 12953114 10.1105/tpc.014183 MAANOVA: A software package for the analysis of spotted cDNA microarray experiments Wu H Kerr MK Cui X Churchill GA The analysis of gene expression data: methods and software Springer-Verlag Parmigiani G, Garrett ES, Irizarry RA, Zeger S 2003 313 431 EST analysis of the Cnidarian <it>Acropora millepora </it>reveals extensive gene loss and rapid sequence divergence in the model invertebrates Kortschak RD Samuel G Saint R Miller DJ Curr Biol 2003 13 2190 2195 10.1016/j.cub.2003.11.030 14680636 Unexpected complexity of the Wnt gene family in a sea anemone Kusserow A Pang K Sturm C Hrouda M Lentfer J Schmidt HA Technau U von Haeseler A Hobmayer B Martindale MQ Holstein TW Nature 2005 433 156 160 10.1038/nature03158 15650739 Comparative gene expression in the symbiotic and aposymbiotic <it>Aiptasia pulchella </it>by expressed sequence tag analysis Kuo J Chen MC Lin CH Fang LS Biochemical and Biophysical Research Communications 2004 318 176 186 10.1016/j.bbrc.2004.03.191 Transcript patterns associated with ectomycorrhiza development in <it>Eucalyptus globules </it>and <it>Pisolithus microcarpus</it> Duplessis S Courty PE Tagu D Martin F New Phytologist 2005 165 599 611 10.1111/j.1469-8137.2004.01248.x 15720670 Molecular and cellular profiles of insect bacteriocytes: mutualism and harm at the initial evolutionary step of symbiogenesis Heddi A Vallier A Anselme C Xin H Rahbe Y Wackers F Cellular Microbiology 2005 7 293 305 15659072 Transcriptome analysis of the aphid bacteriocyte, the symbiotic host cell that harbors an endocellular mutualistic bacterium, <it>Buchnera</it> Nakabachi A Shigenobu S Sakazume N Shiraki T Hayashizaki Y Carninci P Ishikawa H Kudo T Fukatsu T Proc Nat Acad Sci 2005 102 5477 5482 555734 15800043 10.1073/pnas.0409034102 Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host Whitehead LF Douglas AE J Exp Biol 2003 206 3149 3157 10.1242/jeb.00539 12909696 Glycerol Excretion by Symbiotic Algae from Corals and Tridacna and its Control by the Host Muscatine L Science 1967 156 516 519 Fatty acids and hydrocarbons of Caribbean corals Meyers PA Proceedings of the Third International Coral Reef Symposium 1977 1 1529 536 Symbiotic zooxanthellae provide the host-coral <it>Montipora digitata </it>with polyunsaturated fatty acids Papina M Meziane T van Woesik R Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 2003 135 533 537 10.1016/S1096-4959(03)00118-0 Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A Kershaw NJ Mukherji M MacKinnin CH Claridge TD Odell B Wierzbicki AS Lloyd MD Schofield CJ Bioorg Med Chem Lett 2001 11 2545 2548 10.1016/S0960-894X(01)00494-2 11549466 The role of long chain fatty acyl-CoA as signaling molecules in cellular metabolism Black PN Faergeman NJ DiRusso CC Symposium of the American Society for Nutritional Sciences 2000 305S 309S Evidence for the involvement of polyunsaturated fatty acids in the regulation of long-chain acyl CoA thioesterases and peroxisome proliferation in rat carcinosarcoma Ramos KL Colquhoun A Cell Biochem Funct 2001 19 1 9 10.1002/cbf.886 11223865 Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI Nieland TJ Ehrlich M Krieger M Kirchhausen T Biochim Biophys Acta 2005 1734 44 51 15866482 <it>Drosophila </it>RNAi screen reveals CD36 family member required for mycobacterial infection Philips JA Rubin EJ Perrimon N Science 2005 309 1251 1253 10.1126/science.1116006 16020694 The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus Scarselli E Ansuini H Cerino R Roccasecca RM Acali S Filocamo G Traboni C Nicosia A Cortese R Vitelli ATitle Embo Journal 2002 21 5017 5025 129051 12356718 10.1093/emboj/cdf529 High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B Type I Voisset C Callens N Blanchard E De Beeck AO Dubuisson J Vu-Dac N J Biol chem 2005 280 7793 9 15632171 10.1074/jbc.M411600200 Carrageenans inhibit the in vitro growth of <it>Plasmodium falciparum </it>and cytoadhesion to CD36 Adams Y Smith SL Schwartz-Albiez R Andrews KT Parasitol Res 2005 97 290 4 10.1007/s00436-005-1426-3 16012863 Ribosomal RNA sequences of <it>Sarcocystis muris</it>, <it>Theileria annulata</it>, and <it>Crypthecodinium cohnii </it>reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates Gajadhar AA Marquardt WC Hall R Gunderson J Carmona EVA Sogin ML Mol Biochem Parasit 1991 45 147 154 10.1016/0166-6851(91)90036-6 The deep roots of eukaryotes Baldauf SL Science 2003 300 1703 1706 10.1126/science.1085544 12805537 Sphingosine-1-phosphate: An enigmatic signaling lipid Spiegel S Milstien S Nature Reviews Molecular Cell Biology 2003 4 397 407 10.1038/nrm1103 12728273 Generation and metabolism of bioactive sphingosine-1-phosphate Le Stunff H Milstien S Spiegel S Journal of Cellular Biochemistry 2004 92 882 899 10.1002/jcb.20097 15258913 Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-alpha signaling Xia P Wang LJ Moretti PAB Albanese N Chai FG Pitson SM D'Andrea RJ Gamble JR Vadas MA Journal of Biological Chemistry 2002 277 7996 8003 10.1074/jbc.M111423200 11777919 Role of human sphingosine-1-phosphate phosphatase 1 in the regulation of intra- and extracellular sphingosine-1-phosphate levels and cell viability Johnson KR Johnson KY Becker KP Bielawski J Mao CG Obeid LM Journal of Biological Chemistry 2003 278 34541 34547 10.1074/jbc.M301741200 12815058 Sphingosine kinase, sphingosine-1-phosphate, and apoptosis Maceyka M Payne SG Milstien S Spiegel S Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids 2002 1585 193 201 10.1016/S1388-1981(02)00341-4 Manipulation of apoptosis in the host-parasite interaction James ER Green DR Trends in Parasitology 2004 20 280 287 10.1016/j.pt.2004.04.004 15147679 Strategies used by bacterial pathogens to suppress plant defenses Abramovitch RB Martin GB Current Opinion in Plant Biology 2004 7 356 364 10.1016/j.pbi.2004.05.002 15231256 Ceramides modulate programmed cell death in plants Liang H Yao N Song JT Luo S Lu H Greenberg JT Genes Dev 2003 17 2636 41 280613 14563678 10.1101/gad.1140503 A putative coiled-coil domain of prohibitin is sufficient to repress E2F1-mediated transcription and induce apoptosis Joshi B Ko D Ordonez-Ercan D Chellappan SP Biochem Biophys Res Comm 2003 12 459 466 10.1016/j.bbrc.2003.10.148 Expression of prohibitin 3 ' untranslated region suppressor RNA alters morphology and inhibits motility of breast cancer cells Manjeshwar S Lerner MR Zang XP Branam DE Pento JT Lane MM Lightfoot SA Brackett DJ Jupe ER Journal of Molecular histology 2004 5 639 646 Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression Wang S Fusaro G Padmanabhan J Chellappan SP Oncogene 2002 21 8388 8396 10.1038/sj.onc.1205944 12466959 Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling Fusaro G Dasgupta P Rastogi S Joshi B Chellappan S Journal of Biological Chemistry 2003 278 47853 47861 10.1074/jbc.M305171200 14500729 Molecular characterization of two CuZn-superoxide dismutases in a sea anemone Plantivaux A Furla P Zoccola D Garello G Forcioli D Richier S Merle PL Tambutte E Tambutte S Allemand D Free Radical Biology and Medicine 2004 37 1170 1181 10.1016/j.freeradbiomed.2004.06.043 Symbiosis-induced adaptation to oxidative stress Richier S Furla P Plantivaux A Merle PL Allemand D Journal of Experimental Biology 2005 208 277 285 10.1242/jeb.01368 15634847 Oxygen production by endosymbiotic algae controls superoxide dismutase activity in their animal host Dykens JA Shick M Nature 1982 297 579 580 10.1038/297579a0 The winnowing: Establishing the squid – <it>vibrio </it>symbiosis Nyholm SV McFall-Ngai MJ Nat Rev Microbiol 2004 2 632 642 10.1038/nrmicro957 15263898 Biogeography of two species of <it>Symbiodinium </it>(Freudenthal) inhabiting the intertidal sea anemone <it>Anthopleura elegantissima </it>(Brandt) LaJeunesse TC Trench RK Biol Bull 2000 199 126 134 11081711 Symbiont diversity within the widespread scleractinian coral <it>Plesiastrea versipora</it>, across the northwestern Pacific Rodriguez-Lanetty M Hoegh-Guldberg O Mar Biol 2003 143 501 509 10.1007/s00227-003-1105-9 Design considerations for efficient and effective microarray studies Kerr MK Biometrics 2003 59 822 828 10.1111/j.0006-341X.2003.00096.x 14969460 Normalization for cDNA microarray data: A robust composite method adressing single and multiple slide systematic variation Yang YH Dudoit S Lin DM Peng V Ngai J Speed TP Nucleic Acids Research 2002 30 1 e15 99169 11752241 10.1093/nar/30.1.1 Transformation for cDNA microarray data Cui XG Kerr MK Churchill GA Statistical Applications in Genetics and Molecular Biology 2003 2 1 Art. 4 Improved Statistical Tests for Differential Gene Expression by Shrinking Variance Components Cui XG Hwang JTG Qiu J Blades NJ Churchill GA Biostatistics 2005 6 59 75 10.1093/biostatistics/kxh018 15618528 Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data Ramakers C Ruijter JM Deprez RH Moorman AF Neurosci Lett 2003 339 62 66 10.1016/S0304-3940(02)01423-4 12618301 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Genome Biology 2002 3 1 11 10.1186/gb-2002-3-7-research0034