Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation.
Formalin-fixed tissues represent the most abundant clinical material for retrospective studies. However, formalin highly affects macromolecules, impairing their extraction and analysis. In this study, the suitability of some potential substitutes of formalin for RNA-based applications has been considered. Conventional formalin was compared with methacarn and the commercial FineFIX. Their impact on overall RNA preservation was investigated in a cell line-based model fixed during a time course treatment and in a series of fixed human tissues. RNA yield was detected by Nanodrop; ribosomal RNA (rRNA) integrity by electrophoresis and the Agilent Bioanalyzer; messenger RNA (mRNA) integrity by Northern blot and endpoint reverse transcription-polymerase chain reaction; and mRNA amount by real-time polymerase chain reaction. In the cell line model, formalin fixation showed time-dependent detrimental effects on overall RNA preservation. Methacarn and FineFIX were more conservative on both rRNA and mRNA preservation and their impact was time-independent. In tissues, high rRNA degradation levels were found in all fixed specimens, contrasting with the results found in the cells. Conversely, the effects of the fixatives on mRNA integrity reflected the observations shown in the cell line model. In methacarn-fixed samples mRNA amount was also preserved, whereas in formalin and FineFIX-fixed samples it was notably lower when compared with the fresh frozen control. Alcohol-based fixatives are a good solution for long-term fixation of both cytologic and tissue samples by virtue of their time-independent effects on mRNA preservation. In fixed tissue samples, however, the potential effects of preanalytical tissue-related factors should be considered when performing mRNA quantitative analysis.
Dotti
Isabella
I
Dipartimento Universitario Clinico di Scienze Chirurgiche Generali, University of Trieste, Trieste, Italy.
Bonin
Serena
S
Basili
Giorgio
G
Nardon
Ermanno
E
Balani
Alessandro
A
Siracusano
Salvatore
S
Zanconati
Fabrizio
F
Palmisano
Silvia
S
De Manzini
Nicolò
N
Stanta
Giorgio
G
D019342:Acetic Acid / Q000494:pharmacology*; D015152:Blotting, Northern; D002460:Cell Line; D002725:Chloroform / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D000432:Methanol / Q000494:pharmacology*; D057089:Pathology, Molecular / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*
D005404:Fixatives; D012333:RNA, Messenger; D012335:RNA, Ribosomal; C046544:methacarn; D005557:Formaldehyde; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol