Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts

2011 ◽  
Vol 408 (1) ◽  
pp. 169-171 ◽  
Author(s):  
Muriel Filliers ◽  
Ward De Spiegelaere ◽  
Luc Peelman ◽  
Karen Goossens ◽  
Christian Burvenich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Laser Capture

186 LASER CAPTURE MICRODISSECTION FOR GENE EXPRESSION ANALYSIS OF INNER CELL MASS AND TROPHOBLAST FROM BOVINE BLASTOCYSTS

2011 ◽  
Vol 23 (1) ◽  
pp. 194
Author(s):  
M. Filliers ◽  
W. de Spiegelaere ◽  
L. J. Peelman ◽  
K. Goossens ◽  
C. Burvenich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gene Expression Pattern ◽  
Serial Sections ◽  
Trophoblastic Cells ◽  
Keratin 18 ◽  
Laser Capture

Isolation of pure inner cell mass (ICM) and trophoblast samples from a single blastocyst is necessary to obtain accurate information on the transcriptome of these cells. Microsurgical techniques have been described to separate the ICM and trophoblast, but unfortunately, contamination of the ICM cell population with trophoblastic cells is inevitable with these methods. Alternatively, immunosurgery has been described as a valuable technique to obtain a pure ICM sample, although this technique seems to alter the normal gene expression pattern. Laser capture microdissection (LCM) provides the possibility of isolating small tissue fractions from heterogeneous tissue sections, without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. In this study, a protocol is described for the application of LCM to isolate homogeneous ICM and trophoblast samples from single bovine blastocysts for downstream gene expression analysis. The absence of contaminating trophoblastic fractions in the isolated ICM cells was controlled with primers for the keratin 18 (KRT18) gene, which is considered a trophoblast-specific marker in bovine blastocysts. Expanded blastocysts were produced by routine in vitro methods described by (Vandaele et al. 2010 Reproduction 139, 505–511) and fixed in a modified methacarn solution for 24 h. After fixation, the blastocysts were embedded in RNase-free soluble agarose 2%, processed in an STP 420D Tissue Processor, embedded in paraffin, cut in serial sections, and adhered to glass slides, followed by deparaffinization in xylene and staining of the sections with 0.1% cresyl violet in a 85% ethanol solution. Laser capture microdissection was performed as described previously by (De Spiegelaere et al. 2008 Anal. Biochem. 382, 72–74). The ICM was isolated by placing the same cap over 3 to 4 serial sections of one blastocyst. Subsequently, the same procedure was performed with a second cap to isolate the trophoblast. Total RNA was isolated from the LCM-derived ICM and trophoblast on the caps and converted into cDNA. Gene-specific primers for KRT18 (5′-GCAGACCGCTGAGATAGGA-3′ and 5′-GCATATCGGGCCTCCACTT-3′) and for 18S rRNA, a commonly used reference gene (5′-AGAAACGGCTACCACATCCA-3′ and 5′-CACCAGACTTGCCCTCCA-3′), were used and PCR was carried out. Expression of the control gene 18S rRNA was readily detectable in all cell samples. Keratin 18 was detectable in LCM-derived trophoblast, but was absent in the LCM-derived ICM cells, indicative of the successful isolation of ICM cells without contaminating trophoblastic cells. This study demonstrates a novel approach for the application of LCM on small tissue samples that are difficult to handle and which can be used for molecular analysis of specific cell lineages within embryos of different species. Supported by the Fund for Scientific Research–Flanders, Belgium, aspirant 1.1.477.07N00.

Laser capture microdissection protocol for gene expression analysis in the brain

2017 ◽  
Vol 148 (3) ◽  
pp. 299-311 ◽  
Author(s):  
P. Garrido-Gil ◽  
P. Fernandez-Rodríguez ◽  
J. Rodríguez-Pallares ◽  
Jose L. Labandeira-Garcia
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Laser Capture ◽  
The Brain

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection

2018 ◽  
Vol 33 (11) ◽  
pp. 1403-1410 ◽  
Author(s):  
Angela Pucci ◽  
Claudia Mattioli ◽  
Marco Matteucci ◽  
Daniele Lorenzini ◽  
Francesca Panvini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Confocal Microscopy ◽  
Expression Analysis ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Cardiac Myxomas ◽  
Laser Capture

Laser capture microdissection after γ-glutamyl transferase histochemistry: An optimization for gene expression analysis

2014 ◽  
Vol 447 ◽  
pp. 126-132 ◽  
Author(s):  
Julia Esperanza Torres Mena ◽  
Ricardo Sánchez Rodríguez ◽  
Valeria Quintanar Jurado ◽  
Raúl Mojica Espinosa ◽  
Luis Del Pozo Yauner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Glutamyl Transferase ◽  
Laser Capture
Sign in / Sign up

Export Citation Format

Share Document