Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens.
To establish a quantitative method for analysis of gene expressions in small areas of tissue after paraffin embedding, preliminary validation experiments with RT-PCR and Western blotting were performed using methacarn-fixed rodent tissues and a cultured PC12 cell line. A total RNA yield of 52 +/- 15 ng/mm2, sufficient for a quantitative RT-PCR of many genes, could be extracted from a deparaffinized 10-microm-thick rat-liver section by a simple, single-step extraction method. The low concentration of contaminating genomic DNA and the resolution of ribosomal RNAs in RNA gel proved the purity and integrity of the extracted RNA samples, allowing PCR amplification of a long mRNA sequence and mRNA species expressing low copy numbers. PCR amplification of mRNA-derived target gene fragments could be achieved by optimizing the amount of total RNA for reverse transcription and the number of subsequent PCR cycles for each gene. By this validation, organ- and sex-specific mRNA expression could be detected in methacarn-fixed paraffin-embedded tissues without additional DNase treatment of RNA samples. RT-PCR analysis could also be performed with total RNA extracted from deparaffinized tissue dissected with a laser capture microdissection system. In addition, extraction of protein yielded 4.9 +/- 2.1 microg/mm2 from a 10-microm-thick rat-liver section, allowing a quantitative expression analysis of protein by Western blotting. Thus, in addition to its advantages for immunohistochemistry, methacarn-fixed paraffin-embedded tissue has benefits for analysis of both RNAs and proteins in the cells of histologically defined areas.
Shibutani
M
M
Division of Pathology, National Institute of Health Sciences, Kamiyoga, Tokyo, Japan. shibutan@nihs.go.jp
Uneyama
C
C
Miyazaki
K
K
Toyoda
K
K
Hirose
M
M
D019342:Acetic Acid; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002725:Chloroform; D017931:DNA Primers; D005260:Female; D005404:Fixatives; D015870:Gene Expression; D008297:Male; D000432:Methanol; D016716:PC12 Cells; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011916:Rats, Inbred F344; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D005404:Fixatives; C046544:methacarn; D012313:RNA; D002725:Chloroform; D019342:Acetic Acid; D000432:Methanol