Optimization of minuscule samples for use with cDNA microarrays.

@article{citeulike:2470170, abstract = {The recent advent of microarray technology and RNA amplification allows us to compare the expression profiles of thousands of genes from small amounts of tissue or cells. We have compared and contrasted various methods of RNA preparation, RNA amplification, target labelling and array analysis in order to achieve a streamlined protocol for microarraying small samples. We have concluded that usage of the NIA 15K cDNA array set, in combination with RNA extraction using the Mini RNA Isolation kit (Zymo), amplification with the RiboAmp kit (Arcturus), followed by indirect labelling via the Atlastrade mark PowerScripttrade mark Fluorescent Labelling kit (using a modified protocol), is optimal with a material derived from either very early stage mouse embryos or individually picked embryonic stem cell colonies. Normalisation using the analysis package Limma (Bioconductor) with data normalisation by print tip Loess, using the "normexp" function with an offset of 50 for background adjustment, and incorporating A-quantile between array normalisation was best with our results. Furthermore, RT-PCR confirmation of array results is achievable without amplification, thereby controlling for amplification bias. These methods will be of great utility in mapping the transcriptome of embryonic and other small samples.}, address = {School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10, 3US, Wales, UK.}, author = {Hunter, Susan McLean M. and Mansergh, Fiona C C. and Evans, Martin J J. }, citeulike-article-id = {2470170}, doi = {10.1016/j.jprot.2007.11.011}, issn = {0165-022X}, journal = {J Biochem Biophys Methods}, keywords = {amplification, arrays, rna}, month = {December}, posted-at = {2008-05-09 21:43:47}, priority = {4}, title = {Optimization of minuscule samples for use with cDNA microarrays.}, url = {http://dx.doi.org/10.1016/j.jprot.2007.11.011}, year = {2007} }

TY - JOUR ID - citeulike:2470170 TI - Optimization of minuscule samples for use with cDNA microarrays. JF - J Biochem Biophys Methods AD - School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10, 3US, Wales, UK. SN - 0165-022X N2 - The recent advent of microarray technology and RNA amplification allows us to compare the expression profiles of thousands of genes from small amounts of tissue or cells. We have compared and contrasted various methods of RNA preparation, RNA amplification, target labelling and array analysis in order to achieve a streamlined protocol for microarraying small samples. We have concluded that usage of the NIA 15K cDNA array set, in combination with RNA extraction using the Mini RNA Isolation kit (Zymo), amplification with the RiboAmp kit (Arcturus), followed by indirect labelling via the Atlastrade mark PowerScripttrade mark Fluorescent Labelling kit (using a modified protocol), is optimal with a material derived from either very early stage mouse embryos or individually picked embryonic stem cell colonies. Normalisation using the analysis package Limma (Bioconductor) with data normalisation by print tip Loess, using the "normexp" function with an offset of 50 for background adjustment, and incorporating A-quantile between array normalisation was best with our results. Furthermore, RT-PCR confirmation of array results is achievable without amplification, thereby controlling for amplification bias. These methods will be of great utility in mapping the transcriptome of embryonic and other small samples. KW - amplification KW - arrays KW - rna AU - Hunter, Susan McLean AU - Mansergh, Fiona C AU - Evans, Martin J PY - 2007/12/23/ UR - http://dx.doi.org/10.1016/j.jprot.2007.11.011 ER -