An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis.
Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling.
Karsten
Stanislav L
SL
Department of Neurology, Program in Neurogenetics, UCLA School of Medicine, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA.
Van Deerlin
Vivianna M D
VM
Sabatti
Chiara
C
Gill
Lisa H
LH
Geschwind
Daniel H
DH
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D000431:Ethanol / Q000378:metabolism; D005557:Formaldehyde / Q000378:metabolism; D005615:Freezing; D005625:Frontal Lobe / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D005796:Genes; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D051379:Mice; D019579:Neocortex / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013234:Stem Cells / Q000378:metabolism; D014015:Tissue Banks; D016707:Tissue Fixation / Q000379:methods*; D014439:Tyramine / Q000031:analogs & derivatives* / Q000378:metabolism*
D007202:Indicators and Reagents; D012333:RNA, Messenger; D005557:Formaldehyde; D000431:Ethanol; D014439:Tyramine