Reliability and reproducibility of gene expression measurements using amplified RNA from laser-microdissected primary breast tissue with oligonucleotide arrays

Abstract

Combined use of microdissection and high-density oligonucleotide arrays is a powerful technique to study in vivo gene expression. Because microdissection generally yields ng quantities of RNA, RNA amplification is necessary but affects array results. We tested the reliability and reproducibility of oligonucleotide array data obtained from small sample amplified RNA isolated from primary tissues via laser capture microdissection, to determine whether gene expression measurements obtained under these now customary conditions are reliable and reproducible enough to detect authentic expression differences between clinical samples. We performed eight U133A Affymetrix GeneChip oligonucleotide array hybridizations using RNA isolated from a single normal human breast specimen: two standard and six small samples prepared using independent microdissections, RNA isolations, and amplifications. We then performed six array hybridizations using RNA obtained similarly from paired normal epithelium and ductal carcinoma in situ from three independent breast specimens. We determined reliability by analysis of hybridization quality metrics, and reproducibility by analysis of the number of more than twofold changed genes, linear regression, and principal components analysis. All amplified RNA generated good quality hybridizations. From the initial specimen, correlations between replicates (r = 0.96 to 0.99) and between small samples (r = 0.94 to 0.98) were high, and between standard and small samples (r = 0.84) were moderate. In contrast, in the three normal cancer pairs, the differences in gene expression were large among the normal samples, the ductal carcinoma in situ samples, and between normal and ductal carcinoma in situ within each pair. These differences were a much larger source of variability than the technical variability introduced by the processes of laser capture microdissection, small sample amplification, and array hybridization. Nanogram quantities of RNA isolated from primary tissue using laser-capture microdissection generates reliable and reproducible gene expression measurements. These measurements do not mirror those obtained using micrograms of RNA. Biological variability in gene expression between independent specimens, and between histologically distinct samples within a specimen, is greater than the technical variability associated with the procedures. Future studies of in vivo gene expression using this approach will identify functionally important differences within or between specimens.

Figure 1

Schema of tissue acquisition and sample preparation. A: Normal human breast tissue was collected from breast 1 and snap-frozen in liquid nitrogen. In one set of experiments, RNA was directly extracted from pieces of this tissue. Two 5-μg aliquots of RNA (standard A, standard B) were labeled and hybridized according to the standard protocol. Two 100-ng aliquots of RNA (small A, small B) were first amplified and then labeled and hybridized. In the other set of experiments, pieces of snap-frozen tissue were embedded in OCT and sectioned. In small C and small D, the tissue was pulsed with the laser and scraped from the slides (no cap used for collection). In small E and small F, the tissue was acquired as usual, ie, pulsed with the laser through the cap. In small C to small F, RNA was processed in independent amplification, labeling, and hybridization steps. Independent RNA isolations are indicated by a, b, c, and d. Independent first-round amplifications are indicated by 1, 2, 3, 4, and 5. All samples were independently biotin-labeled and hybridized. B: Samples from three different cancer-containing human breast specimens (breast 2, 3, 4) were snap-frozen and embedded in OCT. Paired normal epithelium and DCIS were separately microdissected with caps using the protocol used with small E and small F.

Figure 2

Comparison of hybridization signal intensities. White lines indicate the mean scaled hybridization intensity. Black dots are probesets that change more than twofold between the samples being compared. The fraction of probesets that change more than twofold, and the correlation coefficient (r), are listed at the bottom right of each graph. The left column of scatterplots (a–d) compares hybridization intensity between replicates within each condition. The middle and right columns compare hybridization intensity of individual samples versus the mean intensity of small samples. Each y axis is the log intensity of the single sample indicated. Each x axis is the log mean intensity of all small samples (except for the small sample on the y axis).

Figure 3

Principal components analysis. The two major axes along which the 14 samples vary in gene expression hybridization intensity were determined by principal components analysis. Shown here are the distribution of samples along these two axes, which account for 50.2% of the total observed variation. The scale of each axis is equivalent with respect to the variability represented. Shading reflects tissue type and shape reflects sample protocol. Breast 1 samples are represented in white with black borders; breasts 2, 3, and 4 normal epithelial samples are represented in gray, and cancers are in black. This graph shows that measurements of gene expression from breast 1 small samples, that differ only in the technical details of their processing, are much more similar to each other than are any small samples from breasts 2, 3, or 4, which differ from each other biologically. Each specimen’s normal/cancer pair is joined by an arrow.