Project description:A custom 8x60 k expression microarray for larvae of European fire salamander (Salamandra salamandra) was designed based on transcriptome sequencing. It is known the fact, that oligonucleotide probes differ in the binding behavior towards their target sequences. Therefore, we performed a calibration of our microarray where we assessed the binding behavior of the individual probes empirically. This information was used to normalize gene expression data measurements with the same microarray in another experiment. Please refer to the accompanying publication (Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution) for more information. Labeled cRNA was prepared from Salamander larvae kept at 9°C and 17°C. A cRNA calibration pool was prepared with equimolar amounts of cRNA prepared from (a) a larvae (temperature: 9°C: source: pond KOE), (b) a larvae (temperature: 17°C: source: pond KOE), (c) a larvae (temperature: 9°C: source: stream KoGB (Klufterbach) and (d) a larvae (temperature: 17°C: source: stream KoGB (Klufterbach). See Steinfartz et al. (2007) (doi: 10.1111/j.1365-294X.2007.03490.x) for information of the source populations. Increasing amounts of labeled cRNA (75 ng, 150 ng, 300 ng, 600 ng, 1000 ng, 1400 ng, 1800 ng, 2200 ng), corresponding to (1/8, 1/4, 1/2, 1, 1 2/3, 2 1/3, 3 and 3 3/3 times the recommended amount of 600 ng) were hybridized to 8 microarrays (one microarray per dilution). The change in observed signal intensity in relation to the change in amount of labeled cRNA was used to infer the target-binding behavior of the individual probes. This information was extracted, to be used for a normalization procedure in another experiment with the same microarray (see Czypionka et al. 2015." Ecological transcriptomics – a non-lethal sampling approach for endangered fire salamanders" Methods in Ecology and Evolution). The current study provides only raw data for a calibration experiment, to validate the binding behavior of the different probes on a newly designed microarray for a non model organism (European Fire salamander). This calibration is based only on raw data. More information on targeted genes is provided in a different GEO dataset (currently submitted), where biological meaningful analysis are performed with data which are normalized based on this calibration.

Project description:The analysis of transcriptomes is well-established and increasingly affordable in studies at the interface of ecology and evolution. Expression analysis of thousands of genes in parallel reveals functions and pathways involved in relevant phenotypic differentiation. The application of such methods typically involves the sacrifice of the analysed organisms, which is potentially subject to ethical and legal constraints. As an alternative to lethal sampling, transcriptome analyses can be performed using small biopsies of dispensable tissues. It has to be verified, however, to what extent such results are representative of the whole organism. Here, we use a custom microarray to compare transcriptomes of tail-clip samples with those of the remaining whole-body of fire salamander larvae (Salamandra salamandra). The microarray was calibrated using target RNA to validate the performance of each probe. We varied water temperature to test whether the thermal response in gene expression can be characterized in both types of sample. A large fraction (51 %) of the differentially expressed genes showed parallel changes for both tail clips and whole bodies in response to temperature. While sets of differentially expressed were not identical, they largely belonged to the same functional categories. The gene functions thus revealed a common thermal response of larvae irrespective of the sampled tissue. This included an overexpression of mitochondrial transcripts, an expected thermal acclimatization response of ectotherms. Hence, ecological transcriptomics based on small biopsies represent an alternative to the analysis of lethally sampled tissues in situations where the sacrifice of individuals is not an option. Larvae of European Fire salamander were exposed to two different temperatures (9°C & 17°C; n(cold) = 6, n(warm) = 6, n(total) = 12). The transcriptome response to temperature was assessed based on RNA extracted from tail tips, which can be sampled without sacrificing the individual, and based on RNA extracted from the remaining whole body. Results from the analysis of both tissues were compared.

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