Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.
Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.
Kim
Joung-Ok
JO
Department of Biochemistry, Kyungpook National University, Daegu 700-422, Republic of Korea.
Kim
Hyun-Nam
HN
Hwang
Mi-Hye
MH
Shin
Hong-In
HI
Kim
Shin-Yoon
SY
Park
Rang-Woon
RW
Park
Eui-Yun
EY
Kim
In-San
IS
van Wijnen
Andre J
AJ
Stein
Janet L
JL
Lian
Jane B
JB
Stein
Gary S
GS
Choi
Je-Yong
JY
D019342:Acetic Acid; D000818:Animals; D002725:Chloroform; D004210:Dissection; D005404:Fixatives; D020869:Gene Expression Profiling; D006801:Humans; D007834:Lasers; D000432:Methanol; D051379:Mice; D008813:Mice, Inbred ICR; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
D005404:Fixatives; C046544:methacarn; D012313:RNA; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol