Preservation of RNA for functional genomic studies: a multidisciplinary tumor bank protocol.
Few human tumors are collected such that RNA is preserved for molecular analysis. Completion of the Human Genome Project will soon result in the identification of more than 100,000 new genes. Consequently, increasing attention is being diverted to identifying the function of these newly described genes. Here we describe a multidisciplinary tumor bank procurement protocol that preserves both the integrity of tissue for pathologic diagnosis, and the RNA for molecular analyses. Freshly excised normal skin was obtained from five patients undergoing wound reconstruction following Mohs micrographic surgery for cutaneous neoplasia. Tissues treated for 24 hours with RNAlater were compared histologically and immunohistochemically to tissues not treated with RNAlater. Immunohistochemical stains studied included: CD45, CEA, cytokeratin AE1/3, vimentin, S-100, and CD34 on formalin-fixed, paraffin embedded tissue and CD45 staining of frozen tissue. Slides were blinded and evaluated independently by three pathologists. The histologic and immunohistochemical parameters of tissue stored in RNAlater were indistinguishable from tissue processed in standard fashion with the exception of S-100 stain which failed to identify melanocytes or Langerhan's cells within the epidermis in any of the RNAlater-treated tissues. Interestingly, nerve trunks within the dermis stained appropriately for S-100. Multiple non-cutaneous autopsy tissues were treated with RNAlater, formalin, liquid nitrogen (LN2), and TRIzol Reagent. The pathologists were unable to distinguish between tissues treated with RNAlater, formalin, or frozen in LN2, but could easily distinguish tissues treated with TRIzol Reagent because of extensive cytolysis. RNA was isolated from a portion of the tissue treated with RNAlater and used for molecular studies including Northern blotting and microarray analysis. RNA was adequate for Northern blot analysis and mRNA purified from RNAlater-treated tissues consistently provided excellent templates for reverse transcription and subsequent microarray analysis. We conclude that tissues treated with RNAlater before routine processing are indistinguishable histologically and immunohistochemically from tissues processed in routine fashion and that the RNA isolated from these tissues is of high quality and can be used for molecular studies. Based on this study, we developed a multidisciplinary tumor bank procurement protocol in which fresh tissue from resection specimens are routinely stored in RNAlater at the time of preliminary dissection. Thus, precious human tissue can be utilized for functional genomic studies without compromising the tissue's diagnostic and prognostic qualities.
Department of Dermatology, University of Utah Health Sciences Center, Salt Lake City 84132, USA.
S R
SR
Florell
C M
CM
Coffin
J A
JA
Holden
J W
JW
Zimmermann
J W
JW
Gerwels
B K
BK
Summers
D A
DA
Jones
S A
SA
Leachman
D000368:Aged; D000369:Aged, 80 and over; D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D005089:Exocytosis; D005260:Female; D023281:Genomics; D017408:Guidelines as Topic; D016045:Human Genome Project; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008545:Melanoma / Q000235:genetics / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010340:Pathology, Surgical / Q000379:methods; D012313:RNA / Q000302:isolation & purification; D020871:RNA Stability; D012867:Skin / Q000737:chemistry / Q000166:cytology; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology; D014015:Tissue Banks; D014021:Tissue Preservation / Q000379:methods*
D012313:RNA