Project description:It was recently shown that ultrashort pulse infrared (IR) lasers, operating at the wavelength of the OH vibration stretching band of water, are highly efficient at softly sampling and homogenizing biological tissue. We utilized a nanosecond infrared laser (NIRL) for tissue sampling and homogenization with subsequent LC-MS/MS analysis for mass spectrometric proteomics. Sampling was performed with murine spleen and colon tissue with an ablation volume of 1.1 x 1.1 x0.4 mm³ (approx. 0.5 µl), determined with optical coherence tomography (OCT). The identified proteins of both tissues contained tissue type specific proteins correlating with the corresponding lists of the Human Protein Atlas. The results demonstrate that tissue sampling and homogenization of small tissue volumes less than 1 µL for subsequent mass spectrometric proteomics is feasible with a NIRL and therefore is an alternative towards sampling of tissue sections with laser capture microdissection (LCM).
Project description:To achieve a deep quantitative proteomic profiling of whole tissue with the spatial localization preserved, we designed an untargeted spatial proteomics workflow with a sparse sampling strategy. We combined a computational approach and MS-based proteomics analysis to localize high-throughput proteome mapping within a tissue by microdissecting the slices into a series of parallel strips with different angles, that allow subsequent image reconstruction. We successfully reconstructed a whole-tissue mapping in a mouse brain, consisting of 8,645 protein distributions and expressions with a spatial resolution of approx. 300 μm.
Project description:Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated intra vitam on 43 dogs. Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using phenol-chloroform extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.
Project description:For investigating the molecular physiology and pathophysiology in organs the most exact data will be obtained, if not organ specific cell lines are analysed or the whole organ is homogenized, followed by the analysis of its biomolecules, but if the morphological organisation of the organ can be addressed and in the best case the composition of molecules in single cells of the target organ can be analysed. Laser capture microdissection (LCM) is a technique, which is enabling the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming 2-dimensional technique and optimal results are only obtained, if the tissue is fixed e.g. by formalin. Especially for proteome analysis formalin fixation is reducing the number of identifiable proteins, which is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study the applicability of the infrared laser tissue sampling for proteome analysis of different cell layers of murine intestine was investigated, using LC-MS/MS-based differential quantitative bottom-up proteomics. By laser ablation eight consecutive layers of the colon tissue were obtained and analyzed. Beside a clear distinguishability of protein profiles between ascending, descending and transversal colon, we identified in the different intestinal cell layers proteins, which are cell-specific as confirmed with data from the human protein atlas. Thus, for the first time the sampling directly from intact fresh-frozen tissue with 3-dimensional resolution is giving access to the different proteomes of the different cell layers of colon tissue.
Project description:How piRNA-mediated genome defense achieves specificity against transposons while sampling a complex transcriptome has remained unresolved. Here we show that piRNA biogenesis operates through pervasive, non-specific sampling of cytoplasmic RNAs, with specificity imposed by tissue-specific molecular modules that exploit intrinsic vulnerabilities of transposons. In Drosophila somatic cells, the specificity factor Yb steers basal processing towards uridine-rich RNAs—automatically capturing antisense retrotransposon transcripts due to their intrinsically adenosine-biased genomes. In germline cells lacking Yb, basal sampling generates naïve piRNAs loaded into catalytically active Argonaute proteins, which trigger autocatalytic ping-pong amplification upon encountering complementary targets. In both contexts, transposon mobility facilitates the production of antisense RNAs that enable either biased processing or amplification. Thus, piRNA clusters, long associated with pathway specificity, act as sources of transposon antisense sequences, while specificity arises from layering distinct molecular mechanisms onto a shared foundation of indiscriminate transcript sampling, enabling robust and adaptable genome defense without predefined templates.