Breaking the seals: efficient mRNA detection from human archival paraffin-embedded tissue.
During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under- than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17-19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts.
Illig
Romana
R
Division of Clinical and Functional Anatomy, Innsbruck Medical University, 6020 Innsbruck, Austria.
Fritsch
Helga
H
Schwarzer
Christoph
C
D000328:Adult; D016375:Antisense Elements (Genetics) / Q000235:genetics; D001483:Base Sequence; D000071616:CDX2 Transcription Factor; D004622:Embryo, Mammalian / Q000378:metabolism; D015870:Gene Expression; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012007:Rectum / Q000378:metabolism; D013997:Time Factors; D016707:Tissue Fixation; D014157:Transcription Factors / Q000235:genetics
D016375:Antisense Elements (Genetics); D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; C513184:HOXD13 protein, human; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C099959:homeobox protein HOXA13