Impact of fixative on recovery of mRNA from paraffin-embedded tissue.
Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.
Benchekroun
Mehdi
M
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., San Diego, CA, USA.
DeGraw
Juli
J
Gao
Jane
J
Sun
Li
L
von Boguslawsky
Kristina
K
Leminen
Arto
A
Andersson
Leif C
LC
Heiskala
Marja
M
D019342:Acetic Acid; D000096:Acetone; D000818:Animals; D002725:Chloroform; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D017931:DNA Primers; D000431:Ethanol; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology; D008099:Liver / Q000166:cytology / Q000502:physiology; D000432:Methanol; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000302:isolation & purification*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
C033474:Carnoy's solution; D017931:DNA Primers; D005404:Fixatives; D012333:RNA, Messenger; D000096:Acetone; D005557:Formaldehyde; D000431:Ethanol; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol