Investigating fixative-induced changes in RNA quality and utility by microarray analysis.
Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.
Cox
Melissa L
ML
Drug Safety, Pfizer Global Research and Development, Ann Arbor, MI, USA.
Eddy
Susan M
SM
Stewart
Zachary S
ZS
Kennel
Maggi R
MR
Man
Michael Z
MZ
Paulauskis
Joseph D
JD
Dunstan
Robert W
RW
D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D016477:Artifacts; D002725:Chloroform / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry; D007834:Lasers; D008099:Liver / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D042282:Microdissection; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D018075:RNA, Complementary / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D018075:RNA, Complementary; C046544:methacarn; D005557:Formaldehyde; D000431:Ethanol; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol