March 2013 (No. 1)
Assessment of RNA in human breast tissue sampled by random periareolar ﬁne needle aspiration and ductal lavage and processed as ﬁxed or frozen specimens
Teresa A. Phillips a,b, Carol J. Fabian a,b, Bruce F. Kimler a,c, Brian K. Petroff a,b,*
a Breast Cancer Prevention Center, University of Kansas Medical Center, Kansas City, KS, United States;
b Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS, United States;
c Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS, United States
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.
Reproductive Biology 2013 13 1: 75–81.
* Corresponding author at: Breast Cancer Prevention Center, University of Kansas Medical Center, MS 3003, 3901 Rainbow Boulevard, Kansas City, KS 66160, United States; E-mail address: firstname.lastname@example.org