Preservation of RNA for Functional Genomic Studies: A Multidisciplinary Tumor Bank Protocol

Abstract

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.

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens

“…This is in contrast to other studies certifying RNAlater as an reliable agent not only to stabilise RNA but also to maintain expression profiles. 11,12,23 However, none of these studies included a direct comparison of the expression of single genes between the tested conditions. To our knowledge, there is only one study comparing storage time dependency of gene expression levels using quantitative real-time PCR.…”

“…Purified RNA is susceptible to rapid degradation and must be handled with extreme care. 12,19 However, one should take into consideration that extracted RNA in a test tube and cellular RNA in the cytosolic ambience of an intact cell represent two different settings for a potential degradation process. Histological examination of the Figure 7 Gene expression levels under different storage conditions.…”

“…(2) RNAlater represents a commercially available fixative particularly recommended to preserve RNA and is used in several well-organised tissue biobank projects. 11,12,14 (3) A recent study showed that isotonic saline (0.9% NaCl) provides an alternative to stabilise RNA in tissue samples even at temperatures up to 371C 8 and was therefore included in the study. (4) Since the time after surgical excision ex vivo at room temperature might vary, a time course experiment at room temperature was also performed.…”

“…[8][9][10][11][12][13][14] However, studies are scarce concerning the handling of nonfixed tissue between surgery and subsequent freezing and storage at pathology departments. 15 Immediate freezing of tissue specimens after surgery and further storage at -801C generally serve as the standard procedure for the procurement of surgical specimens.…”

Repuncturing the Renal Biopsy

“…Using these agents, renal tissue and RNA will remain intact at Ϫ20°C for more than 3 yr (unpublished observation). After such storage of the biopsy core, the nephron segments can still be manually microdissected and the tissue remains suitable for both immunohistochemical and gene expression analysis (22,39).…”