Analysis of gene expression at the RNA level often constitutes a first important step in the characterization of a novel gene. In addition, when using marker genes in this type of analysis molecular and cellular changes can be discovered that may occur in mutant and treated tissues. In contrast to immunohistochemical analyses that require a suitable antibody, RNA In situ hybridization can be readily carried out for neariy any gene of interest. Although ISH is a widely used method, the effort to introduce this technique into each of the BCM-IDDRC laboratories would be considerable and very costly. Thus having a core unit that uses existing and highly sophisticated equipment provides not only cost saving but also ensures efficiency and high quality of data. In situ hybridization has been a method used extensively in various fields of developmental biology. The conventional, manual method allows relatively few slides (usually 10 -20) to be processed at once, and the whole experiment can take at least 3 days plus the exposure time (up to several weeks) when radioactively labeled riboprobes are used. The novel aspect of our in situ core is that we use standardized methods for tissue preparation, tissue sectioning, probe preparation, in situ hybridization and data collection. Our robotic ISH is performed in special flow through hybridization chambers that are fitted into temperature controlled chamber racks on the platform of a Tecan pipetting robot. All solutions (including riboprobes) are delivered by the pipetting robot to the hybridization chambers. This allows us to do many more wash steps compared to using a manual set up. We find that a relatively short (6 hrs) hybridization time gives better results than the overnight incubations used with manual ISH. With short hybridizations, we have the capacity to process up to 192 slides in 20 hours. This means during a normal work-week we can process more than 750 slides. We also have a automated microscope that converts the expression patterns on the slides into electronic files that can be send over the internet and also posted on GenePaint.org, the web site shared with our collaborators at the Max Planck Institute in Germany. We have also developed software (GeneDetect) that allows us to quantify in situ results to help with comparison studies for mutant and wild type tissue or treated and untreated tissue. This standardization of methods together with our vast expertise in handling very different types of tissue enable us to perform these ISH experiment in the most efficient way possible and to obtain excellent results for all the different projects. As noted above, we can process 192 slides in one single ISH run which allows the ability to compare gene expression in sections from animals with different genotypes and/or different treatments hybridized under the same conditions, increasing uniformity of results. Clearly, it would be very difficult and time consuming for individual labs to set up ISH as a standard procedure.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Center Core Grants (P30)
Project #
5P30HD024064-23
Application #
8318649
Study Section
Special Emphasis Panel (ZHD1)
Project Start
Project End
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
23
Fiscal Year
2011
Total Cost
$188,735
Indirect Cost
Name
Baylor College of Medicine
Department
Type
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Kho, Jordan; Tian, Xiaoyu; Wong, Wing-Tak et al. (2018) Argininosuccinate Lyase Deficiency Causes an Endothelial-Dependent Form of Hypertension. Am J Hum Genet 103:276-287
Eblimit, Aiden; Zaneveld, Smriti Agrawal; Liu, Wei et al. (2018) NMNAT1 E257K variant, associated with Leber Congenital Amaurosis (LCA9), causes a mild retinal degeneration phenotype. Exp Eye Res 173:32-43
Lanzieri, Tatiana M; Chung, Winnie; Leung, Jessica et al. (2018) Hearing Trajectory in Children with Congenital Cytomegalovirus Infection. Otolaryngol Head Neck Surg 158:736-744
Madan, Simran; Kron, Bettina; Jin, Zixue et al. (2018) Arginase overexpression in neurons and its effect on traumatic brain injury. Mol Genet Metab 125:112-117
De Maio, Antonia; Yalamanchili, Hari Krishna; Adamski, Carolyn J et al. (2018) RBM17 Interacts with U2SURP and CHERP to Regulate Expression and Splicing of RNA-Processing Proteins. Cell Rep 25:726-736.e7
Reeber, Stacey L; Arancillo, Marife; Sillitoe, Roy V (2018) Bergmann Glia are Patterned into Topographic Molecular Zones in the Developing and Adult Mouse Cerebellum. Cerebellum 17:392-403
Gillentine, Madelyn A; Lupo, Philip J; Stankiewicz, Pawel et al. (2018) An estimation of the prevalence of genomic disorders using chromosomal microarray data. J Hum Genet 63:795-801
Abbott, Megan; Jain, Mahim; Pferdehirt, Rachel et al. (2017) Neonatal fractures as a presenting feature of LMOD3-associated congenital myopathy. Am J Med Genet A 173:2789-2794
Jiang, Xiqian; Chen, Jianwei; Baji?, Aleksandar et al. (2017) Quantitative real-time imaging of glutathione. Nat Commun 8:16087
Lim, Joohyun; Grafe, Ingo; Alexander, Stefanie et al. (2017) Genetic causes and mechanisms of Osteogenesis Imperfecta. Bone 102:40-49

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