Profiling and discovery of novel miRNAs from formalin-fixed, paraffin-embedded melanoma and nodal specimens.
Archived formalin-fixed, paraffin-embedded human tumors are widely available and represent a unique source of morphologically defined material. Formalin-fixed, paraffin-embedded tissue is known to contain a wealth of molecular information in the form of microRNAs (miRNAs), which could be correlated with clinical outcome for improved prognostication and/or treatment response. miRNAs are endogenous, noncoding RNAs ( approximately 22 nucleotides) and may function as tumor suppressors or oncogenes. A reliable, robust methodology is needed to take full advantage of archived human cancers, especially for those where fresh-frozen tumor banks are unavailable, for example, malignant melanoma. To this end, we applied a simple-to-use protocol for extracting total RNA from various formalin-fixed, paraffin-embedded specimens (colon, liver, prostate, thyroid, uterus, and skin), optimized for small RNA recovery. Using a "poison primer" strategy (ie, primer silencing), we blocked the amplification of ribosomal RNA, enabling the successful sequencing of 17 novel and 53 known miRNAs (including small RNAs) from 10-year-old archived normal skin, cutaneous scalp melanoma, and sentinel lymph nodes (both negative and positive for metastasis) excised from a 52-year-old man. The cloning incidence provided an estimation of the level of specific miRNA expression, which was confirmed by Northern analysis and quantitative real-time polymerase chain reaction. This methodology can therefore be used to facilitate miRNA discovery from archived human cancers.
Ma
Zhihai
Z
Departments of Pathology, Stanford University School of Medicine,Stanford, California, USA.
Lui
Weng-Onn
WO
Fire
Andrew
A
Dadras
Soheil S
SS
D015152:Blotting, Northern; D002648:Child; D005557:Formaldehyde / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D035683:MicroRNAs / Q000032:analysis / Q000235:genetics*; D008875:Middle Aged; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D016612:Paraffin Embedding / Q000379:methods*; D016133:Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods*
D035683:MicroRNAs; D005557:Formaldehyde