Assessment of RNA in human breast tissue sampled by random periareolar fine needle aspiration and ductal lavage and processed as fixed or frozen specimens.

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) have both been proposed as minimally invasive techniques to sample breast tissue during breast cancer prevention trials. Laser capture microdissection (LCM), linear RNA amplification and quantitative real-time polymerase chain reaction (qPCR) theoretically overcome the limitations of small specimen size obtained with DL and RPFNA. In order to test the yield, relative stability and amplifiability of RNA from fixed and archived RPFNA and DL specimens, breast tissue was sampled from individual high risk women (n=9) by both DL and RPFNA. RPFNA samples showed good RNA/cDNA yield and amplification while only 2 of 9 of the paired DL specimens had cDNA of adequate quality for subsequent PCR. One and two rounds of linear amplification provided approximately a 200- and 20,000-fold enrichment of RNA, respectively. PCR analysis consistently detected ER and COX-1 mRNA in the majority of RPFNA samples examined while pS2, PCNA, VEGF and survivin expression varied with subject. RNA yield and/or stability was greater for fixed and archived RPFNA than DL specimens of breast tissue. In a subsequent study examining an expanded biomarker gene panel in fixed vs. frozen RPFNA samples, mRNA profiles and ranked relative mRNA abundance were similar (r=0.89) for frozen and fixed RPFNA specimens. In summary, frozen RPFNA samples may be optimal for RNA endpoints in human breast cancer prevention trials but fixed RPFNA specimens allow similar analyses with greater convenience.