Optimization of minuscule samples for use with cDNA microarrays.
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 Atlas PowerScript 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.
Hunter
Susan McLean
SM
School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10, 3US, Wales, UK.
Mansergh
Fiona C
FC
Evans
Martin J
MJ
D000818:Animals; D002417:Cattle; D002460:Cell Line; D003851:Deoxyribonucleases / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism; D005784:Gene Amplification / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D012313:RNA; D003851:Deoxyribonucleases