Evaluation of non-formalin tissue fixation for molecular profiling studies.
Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).
Gillespie
John W
JW
Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Rm. 2C500. Bldg. 10, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Best
Carolyn J M
CJ
Bichsel
Verena E
VE
Cole
Kristina A
KA
Greenhut
Susan F
SF
Hewitt
Stephen M
SM
Ahram
Mamoun
M
Gathright
Yvonne B
YB
Merino
Maria J
MJ
Strausberg
Robert L
RL
Epstein
Jonathan I
JI
Hamilton
Stanley R
SR
Gannot
Gallya
G
Baibakova
Galina V
GV
Calvert
Valerie S
VS
Flaig
Michael J
MJ
Chuaqui
Rodrigo F
RF
Herring
Judi C
JC
Pfeifer
John
J
Petricoin
Emmanuel F
EF
Linehan
W Marston
WM
Duray
Paul H
PH
Bova
G Steven
GS
Emmert-Buck
Michael R
MR
D000199:Actins / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000431:Ethanol; D005069:Evaluation Studies as Topic; D005404:Fixatives / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000737:chemistry* / Q000166:cytology; D008297:Male; D016612:Paraffin Embedding; D011467:Prostate / Q000737:chemistry* / Q000166:cytology; D017430:Prostate-Specific Antigen / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D016707:Tissue Fixation / Q000379:methods*
D000199:Actins; D005404:Fixatives; D011506:Proteins; D000431:Ethanol; D012313:RNA; D004247:DNA; D017430:Prostate-Specific Antigen