Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

BACKGROUND: Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdissection (LCM). Our aim was to optimize the isolation of high-quality RNA from laser capture microdissected fresh frozen prostate tissue on the level of staining and RNA extraction. RESULTS: Cresyl violet and haematoxylin were compared as histological stains for LCM. While RNA quality was similar for cresyl violet (median RIN 7.4) and haematoxylin (median RIN 7.6), tissue morphology was more detailed with cresyl violet as compared to haematoxylin. RNA quality from the following kits was compared: RNeasy(®) Micro (median RIN 7.2), miRNeasy Mini (median RIN 6.6), Picopure(®) (median RIN 6.0), mirVana™ miRNA (median RIN 6.5) and RNAqueous(®)-Micro (median RIN 6.3). RNA quality from microdissected samples with either the RNeasy Micro or miRNeasy Mini kit, was comparable to RNA isolated directly from whole tissue slices (median RIN 7.5, p = 0.09). Isolated RNA from benign and prostate cancer microdissected tissue demonstrated that RNA quality can vary between regions from the same clinical sample. Additionally, RNA quality (r = 0.89), but not quantity (r = 0.69) could be precisely measured with the Agilent Bioanalyzer. CONCLUSIONS: We demonstrate that staining with cresyl violet results in the isolation of high quality RNA from laser capture microdissected tissue with high discriminative morphology. The RNeasy Micro and miRNeasy Mini RNA extraction kits generated the highest quality RNA compared to Picopure, mirVana and RNAqueous with minimal loss of RNA quality during LCM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-015-1813-5) contains supplementary material, which is available to authorized users

Are in vitro methods for the detection of endocrine potentials in the aquatic environment predictive for in vivo effects? : Outcomes of the projects SchussenAktiv and SchussenAktivplus in the Lake Constance area, Germany

Many studies about endocrine pollution in the aquatic environment reveal changes in the reproduction system of biota. We analysed endocrine activities in two rivers in Southern Germany using three approaches: (1) chemical analyses, (2) in vitro bioassays, and (3) in vivo investigations in fish and snails. Chemical analyses were based on gas chromatography coupled with mass spectrometry. For in vitro analyses of endocrine potentials in water, sediment, and waste water samples, we used the E-screen assay (human breast cancer cells MCF-7) and reporter gene assays (human cell line HeLa-9903 and MDA-kb2). In addition, we performed reproduction tests with the freshwater mudsnail Potamopyrgus antipodarum to analyse water and sediment samples. We exposed juvenile brown trout (Salmo trutta f. fario) to water downstream of a wastewater outfall (Schussen River) or to water from a reference site (Argen River) to investigate the vitellogenin production. Furthermore, two feral fish species, chub (Leuciscus cephalus) and spirlin (Alburnoides bipunctatus), were caught in both rivers to determine their gonadal maturity and the gonadosomatic index. Chemical analyses provided only little information about endocrine active substances, whereas the in vitro assays revealed endocrine potentials in most of the samples. In addition to endocrine potentials, we also observed toxic potentials (E-screen/reproduction test) in waste water samples, which could interfere with and camouflage endocrine effects. The results of our in vivo tests were mostly in line with the results of the in vitro assays and revealed a consistent reproduction-disrupting (reproduction tests) and an occasional endocrine action (vitellogenin levels) in both investigated rivers, with more pronounced effects for the Schussen river (e.g. a lower gonadosomatic index). We were able to show that biological in vitro assays for endocrine potentials in natural stream water reasonably reflect reproduction and endocrine disruption observed in snails and field-exposed fish, respectively