- Email: [email protected]
Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts
Analytical Biochemistry 408 (2011) 169–171
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts Muriel Filliers a,⇑,1, Ward De Spiegelaere b,1, Luc Peelman c, Karen Goossens c, Christian Burvenich d, Leen Vandaele a, Pieter Cornillie b, Ann Van Soom a a
Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium Department of Nutrition, Genetics, and Ethology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium d Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium b c
a r t i c l e
i n f o
Article history: Received 9 July 2010 Received in revised form 10 August 2010 Accepted 25 August 2010 Available online 16 September 2010
a b s t r a c t Isolation of pure inner cell mass (ICM) and trophectoderm (TE) samples from a single blastocyst is necessary to obtain accurate information on the transcriptomes of these cells. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. However, the small size of blastocysts hampers tissue processing prior to LCM. This article describes a protocol for the application of LCM to isolate homogeneous ICM and TE cell samples from single bovine blastocysts for downstream gene expression analysis. Ó 2010 Elsevier Inc. All rights reserved.
One of the earliest signs of cell differentiation in a preimplantation embryo is evident at the blastocyst stage with the formation of two distinct cell populations. The trophectoderm (trophoblast) (TE)2 consists of outer primitive epithelial cells that enclose the blastocoel and will become the embryonic part of the placenta. The inner cell mass (ICM), composed of a cluster of pluripotent cells out of which all embryonic tissue as well as part of the extraembryonic membranes will develop, is eccentrically concentrated on the inside of the TE layer. Comparing gene expression patterns of ICM and TE cells will contribute to our understanding of these primary steps of cellular differentiation [1]. Isolation of pure homogeneous ICM and TE samples from a single embryo is imperative to obtain accurate information on their transcriptomes. As early as 1972, mechanical dissection strategies were described to separate the two distinct cell populations using microsurgery [2]. More recently, successful laserassisted ICM dissection and subsequent cultivation of embryonic stem cells was demonstrated [3]. Unfortunately, contamination of the ICM cell population with TE cells is inevitable with these methods, and this may substantially bias the results. Alternatively, immunosurgery has been described as a technique to obtain pure ICM, by selective killing of the outer TE cells, to derive embryonic stem cells ⇑ Corresponding author. Fax: +32 9 264 77 97. E-mail address: muriel.fi[email protected] (M. Filliers). These authors contributed equally to this work. 2 Abbreviations used: TE, trophectoderm (trophoblast); ICM, inner cell mass; LCM, laser capture microdissection; PBS, phosphate-buffered saline; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cDNA, complementary DNA; PCR, polymerase chain reaction; rRNA, ribosomal RNA; qPCR, real-time PCR; cChIP, carrier chromatin immunoprecipitation. 1
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.08.032
[4,5] and has been used to depict comparative molecular portraits of ICM and TE cells in human blastocysts [1]. However, during immunosurgery, living blastocysts are exposed to various chemical substances such as oxidizing acids to label the outer cells, and this technique seems to alter the normal gene expression pattern [6]. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells [7–9]. Although LCM with frozen sections is optimal for preserving an adequate RNA quality, the small size of blastocysts impedes tissue handling and sectioning of frozen samples. Therefore, in the current study, a protocol was optimized where bovine blastocysts were embedded in an agarose gel prior to paraffin embedding and sectioning, thereby providing the possibility to specifically isolate ICM and TE cell samples with LCM for downstream gene expression analysis. The absence of contaminating TE fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered to be a trophectodermal marker in both human and bovine blastocysts [1,10,11]. Bovine embryos were produced by routine in vitro methods described by Vandaele and coworkers [12]. Expanded zona-intact blastocysts (day 8 postinsemination) allocated as controls were washed three times in phosphate-buffered saline (PBS) prior to freezing at 80 °C until RNA extraction. Blastocysts for LCM were washed three times in PBS and subsequently fixed in a modified methacarn solution (methanol and acetic acid in an 8:1 ratio) for 24 h. This fixative provides a morphological quality similar to that of formaldehyde fixatives while preserving an adequate RNA
170
Notes & Tips / Anal. Biochem. 408 (2011) 169–171
integrity for molecular analysis [9,13]. After fixation, the blastocysts were embedded in RNase-free soluble agarose (2%) at 60 °C, and this gel was left to polymerize shortly at 4 °C (Sigma–Aldrich, Bornem, Belgium) to enable the subsequent handling steps for paraffin embedding. The agarose gels were processed in an STP 420D Tissue Processor (Microm, Prosan, Merelbeke, Belgium) and paraffin embedded with the embedding center EC 350-1 (Microm). Serial sections of 10 lm were cut with a rotary microtome HM 360 (Microm) and adhered to glass slides with a 0.7% gelatin solution. The sections were deparaffinized in xylene and submerged for 30 s in a bath of 100% and 95% ethanol. Subsequently, the sections were stained with 0.1% cresyl violet in an 85% ethanol solution and dehydrated by submersion for 30 s in 95% and 100% ethanol and two times for 3 min in xylene. This staining protocol avoids processing in aqueous solution, thereby preventing extensive RNA degradation [14]. LCM was performed as described previously [8]. Cells were isolated on Capsure Macro LCM caps (Molecular Devices, Berkshire, UK) using an Arcturus Pixcell IIe LCM device (Molecular Devices). The spot size was minimized by putting the size lever at 7.5 lm and setting the threshold voltage at 190 mV, the laser pulse power at 70 mW, and the pulse width at 0.50 ms. The ICM of one blastocyst was isolated by placing the same cap over three or four serial sections of one blastocyst (Fig. 1). Subsequently, the same procedure was performed with a second cap to isolate the TE cells. During the processing steps and the LCM procedure, RNA degradation was minimized by using RNase-free products only. In addition, all surfaces and recipients were washed with RNase Away (Sigma–Aldrich) and water was ultrapurified with an RNase retention filter using the Modulab Ultra Clear System (Eurowater, Nazareth, Belgium). Total RNA was isolated from both the LCM-derived cells on the caps and the frozen control blastocysts with a PicoPure RNA Isolation Kit (Molecular Devices) according to the manufacturer’s instructions. An in-solution DNase digestion was carried out to remove any contaminating DNA by treating the total RNA with 2 U of RQ1 DNase (Promega, Netherlands), followed by a spin-column purification (Microcon YM-100, Millipore, Belgium). A minus reverse transcription (RT) control with primers for glyceraldehyde3-phosphate dehydrogenase (GAPDH) [15] was performed to ensure that all genomic DNA was properly digested. Due to the minute amount of sample used for RNA extraction, the RNA quantity could not be measured with the BioPhotometer (Eppendorf, Belgium) or the Nanodrop ND 1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). First-strand complementary DNA was synthesized from the RNA with the iScript complementary DNA (cDNA) synthesis kit according to the manufacturer’s instructions (Bio-Rad, Belgium). To verify successful isolation of ICM and TE fractions, gene-specific RT–PCR (polymerase chain reaction) was carried out with samples of LCM-derived ICM and TE samples and on positive controls (i.e., LCM-derived whole blastocysts as well as whole frozen
Fig. 2. GelRed gel (inverted image) of RT–PCR of KRT18 and 18S rRNA. Expression of 18S rRNA was readily detectable in all cell samples. KRT18 was detectable in LCMderived complete blastocysts and LCM-derived TE cells but was absent in the LCMderived ICM cells. Lane 1: control (untreated) blastocysts; lane 2: LCM-derived whole blastocysts; lane 3: LCM-derived TE; lane 4: LCM-derived ICM; lane 5: negative control (blank).
blastocysts). Gene-specific primers for KRT18 were used as a specific marker for TE, and 18S ribosomal RNA (rRNA) was used as a control gene. These primer pairs—for KRT18 (50 -GCAGACCGCTGAG ATAGGA-30 and 50 -GCATATCGGGCCTCCACTT-30 , 169 bp) and for 18S rRNA (50 -AGAAACGGCTACCACATCCA-30 and 50 -CACCAGACTTG CCCTCCA-30 , 144 bp)—were used as described previously [10,14]. PCR was carried out using the following cycling profile: 5 min denaturation at 95 °C followed by 40 cycles of 15 s at 95 °C, 15 s at 62 °C, and 30 s at 72 °C, and a final extension for 10 min at 72 °C. Aliquots of the LCM-derived samples and the positive control blastocysts were separated on a 2% agarose gel and visualized with GelRed (Biotium, VWR, Leuven, Belgium) (Fig. 2). Negative control reactions, containing all reagents except the RNA, were carried out in parallel. Total RNA of untreated whole blastocysts served as a positive control. Expression of the control gene 18S rRNA was readily detectable in all cell samples. KRT18 was detectable in LCM-derived complete blastocysts and LCM-derived TE cells but was absent in the LCMderived ICM cells, indicative of the successful isolation of ICM cells without contaminating TE cells. In conclusion, this proof-of-principle study demonstrates a novel approach to the application of LCM on small tissue samples that are difficult to handle. The pre-embedding in a 2% agarose gel enables subsequent tissue processing and isolation of specific fractions, as demonstrated with the isolation of homogeneous ICM fractions from a single bovine embryo without contamination of TE cells. Although the total amount of RNA isolated with this technique is too small for analyzing large sets of genes, RNA amplification methods that enable cDNA microarrays [16] and large-scale real-time PCR (qPCR) gene expression studies [17] using limited amounts of sample material have been described. The methodology described might be used for molecular analysis of specific cell lineages within embryos of different species, including human. This methodology can easily be used to compare gene expression patterns of exclusively pluripotent or differentiated cell populations from embryos with differences in developmental competence. Furthermore, this isolation technique may be suited for carrier chromatin immunoprecipitation (cChIP) assays, which
Fig. 1. Cresyl violet-stained sections of an expanded zona-intact bovine blastocyst before (A) and after (B) LCM of the ICM fraction. The microdissected zone is attached to the polymer film substrate of the cap (C). ICM cells (white arrowheads) are surrounded by trophectodermal cells (black arrowheads), and the entire blastocyst is encompassed by the zona pellucida (white arrow). Scale bar = 50 lm.
Notes & Tips / Anal. Biochem. 408 (2011) 169–171
allow the epigenetic characterization of distinct subpopulations of cells in the early embryo [18]. Acknowledgments The authors thank Jurgen De Craene, Isabel Lemahieu, and Petra Van Damme for their excellent technical assistance. The authors also thank Paul Simoens for his critical review of the manuscript and constructive comments. The first author (M. Filliers) is a research fellow of the Fund for Scientific Research–Flanders, Belgium (FWO), aspirant 1.1.477.07N00. References [1] J. Adjaye, J. Huntriss, R. Herwig, A. BenKahla, T.C. Brink, C. Wierling, C. Hultschig, D. Groth, M.L. Yaspo, H.M. Picton, R.G. Gosden, H. Lehrach, Primary differentiation in the human blastocyst: Comparative molecular portraits of inner cell mass and trophectoderm cells, Stem Cells 23 (2005) 1514–1525. [2] R.L. Gardner, An investigation of inner cell mass and trophoblast tissues following their isolation from the mouse blastocyst, J. Embryol. Exp. Morphol. 28 (1972) 279–312. [3] N. Tanaka, T. Takeuchi, Q.V. Neri, E.S. Sills, G.D. Palermo, Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: application and preliminary results in a murine model, J. Trans. Med. 4 (2006) 20. [4] D. Solter, B.B. Knowles, Immunosurgery of mouse blastocyst, Proc. Natl. Acad. Sci. USA 72 (1975) 5099–5102. [5] K. Hardy, A.H. Handyside, R.M. Winston, The human blastocyst: cell number death, and allocation during late preimplantation development in vitro, Development 107 (1989) 597–604. [6] G. Cauffman, H. Van de Velde, I. Liebaers, A. Van Steirteghem, DAZL expression in human oocytes, preimplantation embryos, and embryonic stem cells, Mol. Hum. Reprod. 11 (2005) 405–411. [7] M.R. Emmert-Buck, R.F. Bonner, P.D. Smith, R.F. Chuaqui, Z. Zhuang, S.R. Godstein, R.A. Weiss, L.A. Liottan, Laser capture microdissection, Science 274 (1996) 998–1001.
171
[8] W. De Spiegelaere, T. Erkens, J. De Craene, C. Burvenich, L. Peelman, W. Van den Broeck, Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples, Anal. Biochem. 382 (2008) 72–74. [9] M. Cox, S.M. Eddy, Z.S. Stewart, M.R. Kennel, M.Z. Man, J.D. Paulauskis, R.W. Dunstan, Investigating fixative-induced changes in RNA quality and utility by microarray analysis, Exp. Mol. Pathol. 84 (2008) 156–172. [10] J. Adjaye, R. Herwig, T.C. Brink, D. Herrmann, B. Greber, S. Sudheer, D. Groth, J.W. Carnwath, H. Lehrach, H. Niemann, Conserved molecular portraits of bovine and human blastocysts as a consequence of the transition from maternal to embryonic control of gene expression, Physiol. Genomics 31 (2007) 15–27. [11] K. Goossens, A. Van Soom, M. Van Poucke, L. Vandaele, J. Vandesompele, A. Van Zeveren, L.J. Peelman, Identification and expression analysis of genes associated with bovine blastocyst formation, BMC Dev. Biol. 7 (2007) 64. [12] L. Vandaele, M. Thys, J. Bijttebier, A. Van Langendonckt, I. Donnay, D. Maes, E. Meyer, A. Van Soom, Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development, Reproduction 139 (2010) 505–511. [13] W. De Spiegelaere, P. Cornillie, C. Burvenich, W. Van den Broeck, Immunohistochemical detection of hypoxia inducible factors and angiogenic growth factors during endochondral and intramembranous ossification, Anat. Histol. Embryol. 39 (2010) 376–384. [14] M. Clement-Ziza, A. Munnich, S. Lyonnet, F. Jaubert, C. Besmond, Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions, RNA 14 (2008) 2698–2704. [15] K. Goossens, M. Van Poucke, A. Van Soom, J. Vandesompele, A. Van Zeveren, L. Peelman, Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos, BMC Dev. Biol. 5 (2005) 27. [16] L. Hu, J. Wang, K. Baggerly, H. Wang, G.N. Fuller, S.R. Hamilton, K.R. Coombes, W. Zhang, Obtaining reliable information from minute amounts of RNA using cDNA microarrays, BMC Genomics 3 (2002) 16. [17] J. Vermeulen, S. Derveaux, S. Lefever, E. De Smet, K. De Preter, N. Yigit, A. De Paepe, F. Pattyn, F. Speleman, J. Vandesompele, RNA pre-amplification enables large-scale RT–qPCR gene-expression studies on limiting sample amounts, BMC Research Notes 2 (2009) 235. [18] L.P. O’Neill, M.D. VerMilyea, B.M. Turner, Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations, Nat. Genet. 38 (2006) 835–841.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts Muriel Filliers a,⇑,1, Ward De Spiegelaere b,1, Luc Peelman c, Karen Goossens c, Christian Burvenich d, Leen Vandaele a, Pieter Cornillie b, Ann Van Soom a a
Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium Department of Morphology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium Department of Nutrition, Genetics, and Ethology, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium d Department of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium b c
a r t i c l e
i n f o
Article history: Received 9 July 2010 Received in revised form 10 August 2010 Accepted 25 August 2010 Available online 16 September 2010
a b s t r a c t Isolation of pure inner cell mass (ICM) and trophectoderm (TE) samples from a single blastocyst is necessary to obtain accurate information on the transcriptomes of these cells. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. However, the small size of blastocysts hampers tissue processing prior to LCM. This article describes a protocol for the application of LCM to isolate homogeneous ICM and TE cell samples from single bovine blastocysts for downstream gene expression analysis. Ó 2010 Elsevier Inc. All rights reserved.
One of the earliest signs of cell differentiation in a preimplantation embryo is evident at the blastocyst stage with the formation of two distinct cell populations. The trophectoderm (trophoblast) (TE)2 consists of outer primitive epithelial cells that enclose the blastocoel and will become the embryonic part of the placenta. The inner cell mass (ICM), composed of a cluster of pluripotent cells out of which all embryonic tissue as well as part of the extraembryonic membranes will develop, is eccentrically concentrated on the inside of the TE layer. Comparing gene expression patterns of ICM and TE cells will contribute to our understanding of these primary steps of cellular differentiation [1]. Isolation of pure homogeneous ICM and TE samples from a single embryo is imperative to obtain accurate information on their transcriptomes. As early as 1972, mechanical dissection strategies were described to separate the two distinct cell populations using microsurgery [2]. More recently, successful laserassisted ICM dissection and subsequent cultivation of embryonic stem cells was demonstrated [3]. Unfortunately, contamination of the ICM cell population with TE cells is inevitable with these methods, and this may substantially bias the results. Alternatively, immunosurgery has been described as a technique to obtain pure ICM, by selective killing of the outer TE cells, to derive embryonic stem cells ⇑ Corresponding author. Fax: +32 9 264 77 97. E-mail address: muriel.fi[email protected] (M. Filliers). These authors contributed equally to this work. 2 Abbreviations used: TE, trophectoderm (trophoblast); ICM, inner cell mass; LCM, laser capture microdissection; PBS, phosphate-buffered saline; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cDNA, complementary DNA; PCR, polymerase chain reaction; rRNA, ribosomal RNA; qPCR, real-time PCR; cChIP, carrier chromatin immunoprecipitation. 1
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.08.032
[4,5] and has been used to depict comparative molecular portraits of ICM and TE cells in human blastocysts [1]. However, during immunosurgery, living blastocysts are exposed to various chemical substances such as oxidizing acids to label the outer cells, and this technique seems to alter the normal gene expression pattern [6]. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells [7–9]. Although LCM with frozen sections is optimal for preserving an adequate RNA quality, the small size of blastocysts impedes tissue handling and sectioning of frozen samples. Therefore, in the current study, a protocol was optimized where bovine blastocysts were embedded in an agarose gel prior to paraffin embedding and sectioning, thereby providing the possibility to specifically isolate ICM and TE cell samples with LCM for downstream gene expression analysis. The absence of contaminating TE fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered to be a trophectodermal marker in both human and bovine blastocysts [1,10,11]. Bovine embryos were produced by routine in vitro methods described by Vandaele and coworkers [12]. Expanded zona-intact blastocysts (day 8 postinsemination) allocated as controls were washed three times in phosphate-buffered saline (PBS) prior to freezing at 80 °C until RNA extraction. Blastocysts for LCM were washed three times in PBS and subsequently fixed in a modified methacarn solution (methanol and acetic acid in an 8:1 ratio) for 24 h. This fixative provides a morphological quality similar to that of formaldehyde fixatives while preserving an adequate RNA
170
Notes & Tips / Anal. Biochem. 408 (2011) 169–171
integrity for molecular analysis [9,13]. After fixation, the blastocysts were embedded in RNase-free soluble agarose (2%) at 60 °C, and this gel was left to polymerize shortly at 4 °C (Sigma–Aldrich, Bornem, Belgium) to enable the subsequent handling steps for paraffin embedding. The agarose gels were processed in an STP 420D Tissue Processor (Microm, Prosan, Merelbeke, Belgium) and paraffin embedded with the embedding center EC 350-1 (Microm). Serial sections of 10 lm were cut with a rotary microtome HM 360 (Microm) and adhered to glass slides with a 0.7% gelatin solution. The sections were deparaffinized in xylene and submerged for 30 s in a bath of 100% and 95% ethanol. Subsequently, the sections were stained with 0.1% cresyl violet in an 85% ethanol solution and dehydrated by submersion for 30 s in 95% and 100% ethanol and two times for 3 min in xylene. This staining protocol avoids processing in aqueous solution, thereby preventing extensive RNA degradation [14]. LCM was performed as described previously [8]. Cells were isolated on Capsure Macro LCM caps (Molecular Devices, Berkshire, UK) using an Arcturus Pixcell IIe LCM device (Molecular Devices). The spot size was minimized by putting the size lever at 7.5 lm and setting the threshold voltage at 190 mV, the laser pulse power at 70 mW, and the pulse width at 0.50 ms. The ICM of one blastocyst was isolated by placing the same cap over three or four serial sections of one blastocyst (Fig. 1). Subsequently, the same procedure was performed with a second cap to isolate the TE cells. During the processing steps and the LCM procedure, RNA degradation was minimized by using RNase-free products only. In addition, all surfaces and recipients were washed with RNase Away (Sigma–Aldrich) and water was ultrapurified with an RNase retention filter using the Modulab Ultra Clear System (Eurowater, Nazareth, Belgium). Total RNA was isolated from both the LCM-derived cells on the caps and the frozen control blastocysts with a PicoPure RNA Isolation Kit (Molecular Devices) according to the manufacturer’s instructions. An in-solution DNase digestion was carried out to remove any contaminating DNA by treating the total RNA with 2 U of RQ1 DNase (Promega, Netherlands), followed by a spin-column purification (Microcon YM-100, Millipore, Belgium). A minus reverse transcription (RT) control with primers for glyceraldehyde3-phosphate dehydrogenase (GAPDH) [15] was performed to ensure that all genomic DNA was properly digested. Due to the minute amount of sample used for RNA extraction, the RNA quantity could not be measured with the BioPhotometer (Eppendorf, Belgium) or the Nanodrop ND 1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). First-strand complementary DNA was synthesized from the RNA with the iScript complementary DNA (cDNA) synthesis kit according to the manufacturer’s instructions (Bio-Rad, Belgium). To verify successful isolation of ICM and TE fractions, gene-specific RT–PCR (polymerase chain reaction) was carried out with samples of LCM-derived ICM and TE samples and on positive controls (i.e., LCM-derived whole blastocysts as well as whole frozen
Fig. 2. GelRed gel (inverted image) of RT–PCR of KRT18 and 18S rRNA. Expression of 18S rRNA was readily detectable in all cell samples. KRT18 was detectable in LCMderived complete blastocysts and LCM-derived TE cells but was absent in the LCMderived ICM cells. Lane 1: control (untreated) blastocysts; lane 2: LCM-derived whole blastocysts; lane 3: LCM-derived TE; lane 4: LCM-derived ICM; lane 5: negative control (blank).
blastocysts). Gene-specific primers for KRT18 were used as a specific marker for TE, and 18S ribosomal RNA (rRNA) was used as a control gene. These primer pairs—for KRT18 (50 -GCAGACCGCTGAG ATAGGA-30 and 50 -GCATATCGGGCCTCCACTT-30 , 169 bp) and for 18S rRNA (50 -AGAAACGGCTACCACATCCA-30 and 50 -CACCAGACTTG CCCTCCA-30 , 144 bp)—were used as described previously [10,14]. PCR was carried out using the following cycling profile: 5 min denaturation at 95 °C followed by 40 cycles of 15 s at 95 °C, 15 s at 62 °C, and 30 s at 72 °C, and a final extension for 10 min at 72 °C. Aliquots of the LCM-derived samples and the positive control blastocysts were separated on a 2% agarose gel and visualized with GelRed (Biotium, VWR, Leuven, Belgium) (Fig. 2). Negative control reactions, containing all reagents except the RNA, were carried out in parallel. Total RNA of untreated whole blastocysts served as a positive control. Expression of the control gene 18S rRNA was readily detectable in all cell samples. KRT18 was detectable in LCM-derived complete blastocysts and LCM-derived TE cells but was absent in the LCMderived ICM cells, indicative of the successful isolation of ICM cells without contaminating TE cells. In conclusion, this proof-of-principle study demonstrates a novel approach to the application of LCM on small tissue samples that are difficult to handle. The pre-embedding in a 2% agarose gel enables subsequent tissue processing and isolation of specific fractions, as demonstrated with the isolation of homogeneous ICM fractions from a single bovine embryo without contamination of TE cells. Although the total amount of RNA isolated with this technique is too small for analyzing large sets of genes, RNA amplification methods that enable cDNA microarrays [16] and large-scale real-time PCR (qPCR) gene expression studies [17] using limited amounts of sample material have been described. The methodology described might be used for molecular analysis of specific cell lineages within embryos of different species, including human. This methodology can easily be used to compare gene expression patterns of exclusively pluripotent or differentiated cell populations from embryos with differences in developmental competence. Furthermore, this isolation technique may be suited for carrier chromatin immunoprecipitation (cChIP) assays, which
Fig. 1. Cresyl violet-stained sections of an expanded zona-intact bovine blastocyst before (A) and after (B) LCM of the ICM fraction. The microdissected zone is attached to the polymer film substrate of the cap (C). ICM cells (white arrowheads) are surrounded by trophectodermal cells (black arrowheads), and the entire blastocyst is encompassed by the zona pellucida (white arrow). Scale bar = 50 lm.
Notes & Tips / Anal. Biochem. 408 (2011) 169–171
allow the epigenetic characterization of distinct subpopulations of cells in the early embryo [18]. Acknowledgments The authors thank Jurgen De Craene, Isabel Lemahieu, and Petra Van Damme for their excellent technical assistance. The authors also thank Paul Simoens for his critical review of the manuscript and constructive comments. The first author (M. Filliers) is a research fellow of the Fund for Scientific Research–Flanders, Belgium (FWO), aspirant 1.1.477.07N00. References [1] J. Adjaye, J. Huntriss, R. Herwig, A. BenKahla, T.C. Brink, C. Wierling, C. Hultschig, D. Groth, M.L. Yaspo, H.M. Picton, R.G. Gosden, H. Lehrach, Primary differentiation in the human blastocyst: Comparative molecular portraits of inner cell mass and trophectoderm cells, Stem Cells 23 (2005) 1514–1525. [2] R.L. Gardner, An investigation of inner cell mass and trophoblast tissues following their isolation from the mouse blastocyst, J. Embryol. Exp. Morphol. 28 (1972) 279–312. [3] N. Tanaka, T. Takeuchi, Q.V. Neri, E.S. Sills, G.D. Palermo, Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: application and preliminary results in a murine model, J. Trans. Med. 4 (2006) 20. [4] D. Solter, B.B. Knowles, Immunosurgery of mouse blastocyst, Proc. Natl. Acad. Sci. USA 72 (1975) 5099–5102. [5] K. Hardy, A.H. Handyside, R.M. Winston, The human blastocyst: cell number death, and allocation during late preimplantation development in vitro, Development 107 (1989) 597–604. [6] G. Cauffman, H. Van de Velde, I. Liebaers, A. Van Steirteghem, DAZL expression in human oocytes, preimplantation embryos, and embryonic stem cells, Mol. Hum. Reprod. 11 (2005) 405–411. [7] M.R. Emmert-Buck, R.F. Bonner, P.D. Smith, R.F. Chuaqui, Z. Zhuang, S.R. Godstein, R.A. Weiss, L.A. Liottan, Laser capture microdissection, Science 274 (1996) 998–1001.
171
[8] W. De Spiegelaere, T. Erkens, J. De Craene, C. Burvenich, L. Peelman, W. Van den Broeck, Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples, Anal. Biochem. 382 (2008) 72–74. [9] M. Cox, S.M. Eddy, Z.S. Stewart, M.R. Kennel, M.Z. Man, J.D. Paulauskis, R.W. Dunstan, Investigating fixative-induced changes in RNA quality and utility by microarray analysis, Exp. Mol. Pathol. 84 (2008) 156–172. [10] J. Adjaye, R. Herwig, T.C. Brink, D. Herrmann, B. Greber, S. Sudheer, D. Groth, J.W. Carnwath, H. Lehrach, H. Niemann, Conserved molecular portraits of bovine and human blastocysts as a consequence of the transition from maternal to embryonic control of gene expression, Physiol. Genomics 31 (2007) 15–27. [11] K. Goossens, A. Van Soom, M. Van Poucke, L. Vandaele, J. Vandesompele, A. Van Zeveren, L.J. Peelman, Identification and expression analysis of genes associated with bovine blastocyst formation, BMC Dev. Biol. 7 (2007) 64. [12] L. Vandaele, M. Thys, J. Bijttebier, A. Van Langendonckt, I. Donnay, D. Maes, E. Meyer, A. Van Soom, Short-term exposure to hydrogen peroxide during oocyte maturation improves bovine embryo development, Reproduction 139 (2010) 505–511. [13] W. De Spiegelaere, P. Cornillie, C. Burvenich, W. Van den Broeck, Immunohistochemical detection of hypoxia inducible factors and angiogenic growth factors during endochondral and intramembranous ossification, Anat. Histol. Embryol. 39 (2010) 376–384. [14] M. Clement-Ziza, A. Munnich, S. Lyonnet, F. Jaubert, C. Besmond, Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions, RNA 14 (2008) 2698–2704. [15] K. Goossens, M. Van Poucke, A. Van Soom, J. Vandesompele, A. Van Zeveren, L. Peelman, Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos, BMC Dev. Biol. 5 (2005) 27. [16] L. Hu, J. Wang, K. Baggerly, H. Wang, G.N. Fuller, S.R. Hamilton, K.R. Coombes, W. Zhang, Obtaining reliable information from minute amounts of RNA using cDNA microarrays, BMC Genomics 3 (2002) 16. [17] J. Vermeulen, S. Derveaux, S. Lefever, E. De Smet, K. De Preter, N. Yigit, A. De Paepe, F. Pattyn, F. Speleman, J. Vandesompele, RNA pre-amplification enables large-scale RT–qPCR gene-expression studies on limiting sample amounts, BMC Research Notes 2 (2009) 235. [18] L.P. O’Neill, M.D. VerMilyea, B.M. Turner, Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations, Nat. Genet. 38 (2006) 835–841.