Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity.
Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.
Cox
Melissa L
ML
Pfizer Global Research and Development, Safety Sciences Ann Arbor, 2800 Plymouth Road, Tox 35/134, Ann Arbor, MI 48105, USA. melissa.cox@pfizer.com
Schray
Carrie L
CL
Luster
Chandra N
CN
Stewart
Zachary S
ZS
Korytko
Peter J
PJ
M Khan
Kanwar Nasir
KN
Paulauskis
Joseph D
JD
Dunstan
Robert W
RW
D000818:Animals; D005404:Fixatives / Q000737:chemistry* / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D012337:RNA, Ribosomal, 18S