Ward De Spiegelaere 1 , Karen Goossens 2 , Mieke Stevens 3 , Ann Van Soom 4 , Luc Peelman 2 , Christian Burvenich 3 , Bart De Spiegeleer 5 , Pieter Cornillie 6 1) Department of General Internal Medicine, Infectious Diseases, and Psychosomatic Medicine 2) Department of Nutrition, Genetics, and Ethology, 3) Department of Physiology and Biometrics, 4) Department of Reproduction, Obstetrics, and Herd Health, 5) Drug Quality and Registration Group, Department of Pharmaceutical Analysis 6) Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection Introduction: The comparison of gene expression profiles between specific cell fractions in early embryos may increase our insight into pathways of initial development. As an example, the seperate isolation and analysis of inner cell mass cells (ICM) and trophoblast cells (TB) from single blastocyst will provide more insight into the molecular pathways triggering cellular differentiation and cell fate development. Tissue processing and microdissection: Blastocysts were produced by coincubating bovine cumulus- oocyte-complexes with bovine sperm, followed by in vitro embryo culture. On day 8, blastocysts with an intact zona pelucida were washed in phosphate buffered saline and fixed in modified methacarn (8 parts methanol and 1 part acetic acid). Conclusion: The present method to isolate specific cellular populations from small embryonic tissues provides the possibility to quantitatively assess differential gene expression between the different cell populations. This tool will help future research to explore the molecular pathways behind pluripotency and stem cell differentiation. Analysis of the quality of the isolates: To validate this procedure for quantitative analysis of RNA molecules it is important to avoid contamination of TB fractions in ICM isolates and vice versa. In addition, RNA degradation should be kept at a minimum. XXIX th Congress of the European Association of Veterinary Anatomists, 25 - 28 July 2012, Stara Zagora, Bulgaria Ward.DeSpiegelaere@UGent.be Applications: The presented method has been successfully applied with quantitative PCR for the quantification of mRNA and micro RNA (miRNA) (Fig 5). Current work is ongoing to optimize a procedure for deep sequencing to allow a comparison of the whole transcriptome between ICM and TB fractions. RNA quality Due to the low amount of isolated RNA, the RNA quality could not be assessed with automated electrophoresis. Consequently, two PCR based assays to investigate RNA quality were used (Fig. 4). These revealed minimal influence of RNA quality on RT-qPCR. Acknowledgements: The authors wish to thank Lobke De Bels and Jurgen De Craene for technical support and Prof. Wim Van den Broeck and Prof. Paul Simoens for scientific advice. References: Goossens et al., Anal Biochem, 2012;423:93-101; De Spiegelaere et al., Methods Mol Biol. 2012;853:29-37; Filliers et al., Anal Biochem, 2011;408:169-171 Here, we describe an optimized protocol for the isolation of ICM and TB cells from bovine blastocysts using Laser capture microdissection (LCM). Histological procedures are hampered by the small size of blastocysts (ᴓ: 100μm), and initial results revealed that cryosectioning of blastocysts is not feasible. Consequently, a procedure was optimized for paraffin embedding of blastocysts prior to microdissection. Fig. 1: Micrograph of a section through a blastocyst stained with haematoxilin and eosin after consecutive agarose and paraffin embedding, performed as described below. After 6 hours fixation, the blastocysts were collected and suspended in a 2% agarose solution at >60°C. This step embeds the blastocysts in a mold that allows paraffin embedding and facilitates manual handling. Serial section of 10 μm were cut, and stained with 0.1% cresyl violet solution in 85% ethanol, to ease visual recognition of the blastocysts while minimizing RNA decay. Finally, the ICM and TB fractions of the blastocysts were isolated using laser capture microdissection Fig. 2: A-D, Illustration of the fixation (A), processing (B), sectioning (C), and microdissection (D) of blastocysts. E-F, Micrographs of a blastocyst before LCM showing the ICM (white arrow) and the TB cells (black arrows; E), after LCM the ICM is removed (F), and the captured ICM is found on the cap (G). A Purity of the isolates Keratin 18 (KRT18) is specifically expressed in TB cells and was used as a marker for contamination of ICM isolates. Minimal to no expression of KRT18 was detected in ICM isolates, indicating neglectable contamination of ICM isolates (Fig. 3) Fig. 3: Agarose gel with RT-PCR products show negative and low positive reactions for KRT18 in ICM, and purely positive in TB isolates (A). Normalized data of all samples reveal only minimal contamination in some ICM isolates (B). Formalin fixation leads to lower KRT18 expression compared to Methacarn fixation (C), pointing to a higher RNA degeneration in formalin fixed samples. Fig. 4: Comparison of different amplicon lengths revealed no significant difference of RT-qPCR quantification of a beta actin primer of 253 bp versus one of 92 bp (A). No difference was observed between primers located at the 3’ versus 5’ end of mRNA for alpha 2 microglobulin (A2M) (B). Fig. 5: A comparison of FISH staining for Bta-miR-155 expression (A-C) with RT-qPCR of microdissected ICM and TB fractions for BTa-miR-155 (D) reveals that this miRNA is mainly expressed in ICM fractions. The combinations of FISH and LCM allows a visual evaluation with a quantitative analysis respectively. B C D E F G A B C A 0 2 4 6 8 10 12 ICM TB Relative KRT18 RNA expression in ICM versus TB B 0 5 10 15 20 25 Formol Methacarn Relative KRT18 expression in formol versus methacarn TB samples C 0 10 20 30 40 50 60 70 80 90 100 ICM TB Relative Bta-miR-155 expression Total Formol Methacarn D 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 Total ICM TB Relative ACTB expression: influence of amplicon lenght ACTB 253 bp ACTB 92 bp A 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 Total ICM TB Relative A2M expression: influence of primer position A2M 5' A2M 3' B