| First Author | Freeman TC | Year | 1998 |
| Journal | MGI Direct Data Submission | Mgi Jnum | J:46439 |
| Mgi Id | MGI:1199209 | Citation | Freeman TC, et al. (1998) Expression Mapping of Mouse Genes. MGI Direct Data Submission |
| abstractText | Summary Tom C. Freeman1, Alistair K. Dixon1, Elizabeth A. Campbell1, Tere-Michelle Tait1, Peter J. Richardson1,2, Kate M. Rice1, Gareth L. Maslen1, Anthony D. Metcalfe3, Charles H. Streuli3, David R. Bentley1. 1. The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambs., CB10 1SA. UK 2. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK 3. School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT,UK We wished to establish feasibility of mapping systematically the expression of a large number of genes as an essential aid to their functional characterisation. The mouse was selected for this pilot study due to its popularity as a genetic model, andthe availability of gene sequence and tissue. A wide range of tissues were obtained and cDNA prepared from total RNA. PCR assays were developed to 3' gene sequences from the public databases. To date, this panel has been used to determine tissue-specific expression profiles of 517 genes across 46 tissues. In one tissue (the mammary gland), we also examined the temporal expression profiles of 44 genes during differentiation (data not included). A wide range of expression patterns was observed, including examples of genes with tissue-independent profiles and genes with very specific profiles that reflect the functional role of the gene product. The methodology has been demonstrated to be robust, sensitive and capable of providing an indication of therelative expression level of each gene across the various tissue types. With the recent rapid increase in available mammalian gene sequences, particularly as a result of the expressed sequence tag (EST) sequencing projects, the sequence of most mammalian genes will soon be known. We propose that this model could be extended to develop a co-ordinated approach to the generation of databases of gene expression. Methods RNA extraction and reverse transcription Tissues from 2 - 10 male and/or female, 6-8week old C57BL/6 mice were pooled and snap frozen. Total RNA was extracted using the RNAce DNA-free total RNA extraction kit (BioExpress, London, UK). 50 ug total RNA was DNase1 digested (0.1 U/ug, 30 min), heated for 2 min at 90 degrees C and chilled on ice. cDNA synthesis was performed in the presence of 20 uM anchored T-primer, T17(AGC) and MMLV reverse transcriptase (Life Technologies) according to the manufacturer's recommendations. The amount of cDNA required for each reaction was standardisedso that the level of ribosomal protein S29 gene PCR product was the same in each sample. This was chosen as a representative gene that is expressed at a comparable level in all tissues and the results are therefore relative to this standard. SufficientcDNA stocks were prepared for the entire study and stored at -20 degrees C in multiple aliquots containing 0.2% glycogen as carrier. Primer design and PCR PCR primers of 20 plus/minus 2 bp in length with a 20-80% GC content and Tm of 60 degrees C plus/minus 3 degrees C were selected using the program PRIMER (Whitehead Institute, Cambridge, MA) to amplify products of 120-190 bp in length from within 300 bp of the 3' end of the sequence. Primers were pre-screened to determine the optimal conditionsfor specific cDNA amplification on 7 pools of cDNAs in the full expression profiling panel, plus mouse genomic DNA. They were tested using 35 and 45 PCR cycles at 55 degrees C annealing temperature under standard assay conditions (see below), but if this failed to give a single band of the predicted size a second pre-screen was performed using 40 and 50 cycles at 60 degrees C annealing temperature. Hot-lid PCR amplification of cDNA equivalent to 10 ng of total RNA was carried out in 1x PCR buffer (3.5mMMgCl2,pH 8.8) containing, 12.5% sucrose, 0.1 mM cresol red, 12 mM beta-mercaptoethanol, 0.5 mM dNTPs (Pharmacia, Milton Keynes, UK), 0.6 U AmpliTaq DNA polymerase (Applied Biosystems, Warrington, UK), and primers were used at 100 ng/reaction. Amplifications were carried out on PTC-225 thermal cyclers (Tetrad, MJ Research, US). Following an initial 2 min denaturing step (92 degrees C), each PCR cycle consisted of 30 sec denaturing (92 degrees C), 90 sec annealing (55 or 60 degrees C), and 60 sec elongation (72degrees C).In a small number of assays the amount of cDNA was increased to 50 or 100 ng per well, or the concentration of primer decreased, to improve the signal. Otherwise all assays were conducted under identical conditions and only the number of cycles or annealing temperature varied. After the final cycle, the reaction was held for 10 min at 72 degrees C. The PCR products were then separated on a 2.5% agarose gel, stained with ethidium bromide and photographed. Figures Each figure shows the results of a single RT-PCR expression profiling assay. Duplicate reactions performed on the cDNA panel are shown one above the other. To aid comparison of expression levels across tissues, the amount of PCR product generated has been indicated using arbitrary scoring system: dark blue - comparatively strong signal in both duplicates; light blue - both duplicates positive with moderate to weak signal; green - weak signal in one of the duplicates; red - relatively strong signal in one duplicatebut not other, indicating a discrepancy in the amplification procedure or experimental error; yellow - no signal in either duplicate. Experimental conditions are shown to the right of the results. Disclaimer and copyright notice If an expression figure is reproducedin a publication, the figure legend should include an explicit statement concerning the source of the information. In most instances, expression data has been generated by a single experiment using a single primer pair with no prior knowledge of the expected expression pattern. We recommend therefore, that users might wish to carry out appropriate confirmatory checks. If you have any questions regarding the expression data or their use in publications, please contact Tom Freeman (tom.freeman@roslin.ed.ac.uk). The Sanger Centre provides these data in good faith, but makes no warranty, express or implied, nor assumes any legal liability or responsibility for any purpose for which the data are used. |
