Effect of one-step 100% ethanol fixation and modified manual microdissection on high-quality RNA recovery from esophageal carcinoma specimen.
This paper attempts to determine an optimal fixation protocol for stabilizing RNA during microdissection so as to obtain high-quality RNA from specific cell populations procured from esophageal carcinoma specimens, and to develop a manual microdissection that can facilitate the procurement. The special features of our protocol include one-step dehydration of tissue sections in 100% ethanol immediately after cryosectioning, a self-made T-shape plate (T plate) and "exclusion microdissection" procedure. The quality of RNA isolated from dissected cells was analyzed by neutral agarose gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) to detect genes of different abundance levels. One-step 100% ethanol fixation of cryosections effectively stabilized RNA integrity for agelong period of time while maintaining histological morphology comparable to that using the conventional procedure, indicating that it is a valid protocol for preservation of RNA in microdissected samples. In conjunction with the application of the T plate and 'exclusion microdissection' procedure, which efficiently simplifies manual microdissection procedure, allowing maximal procurement of target cells from complex primary tissues, full use of every single specimen for maximal procurement of target cells from the sections was allowed. The RNA isolated from 5 different stage-specific cell populations of an esophageal carcinoma specimen was of high quality and sufficient in quantity for various downstream molecular analyses. Our method is suitable for a wide spectrum of expression analysis in diverse clinical settings.
Hu
S-P
SP
Center for Molecular Biology, Shantou University Medical College, Shantou, Guangdong Province, China. sphu@stu.edu.cn
Yang
J-S
JS
Wu
M-Y
MY
Shen
Z-Y
ZY
Zhang
K-H
KH
Liu
J-W
JW
Guan
B
B
D004587:Electrophoresis, Agar Gel; D004938:Esophageal Neoplasms / Q000473:pathology*; D000431:Ethanol / Q000494:pharmacology; D005404:Fixatives / Q000494:pharmacology; D006801:Humans; D042282:Microdissection; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*
D005404:Fixatives; D000431:Ethanol; D012313:RNA