| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Technical Advance |
From the Department of Pathology and Immunology and the Alvin J. Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
In this report, we demonstrate how a modest effort using LCM can yield nanogram quantities of high-quality RNA. To increase standardization across clinical specimens, isolated RNA is quantitated and qualitatively assessed using microcapillary electrophoresis, and staggered internal control transcripts of defined copy number are added to the sample before first-strand cDNA synthesis. Using two rounds of linear amplification, a sufficient quantity of labeled target is generated to hybridize to high-density oligonucleotide arrays (Affymetrix GeneChips; Affymetrix, Santa Clara, CA). Based on expression levels of internal controls, we demonstrate that this procedure is reproducible and results in an analytical sensitivity and precision comparable to standard methodologies that routine use 200 to 1000 times greater input RNA. Using this approach, expression profiling was performed on RNA isolated from nonmalignant mammary ductal epithelial cells and adjacent regions of ductal carcinoma in situ from three patients with breast cancer. Several genes previously implicated in breast cancer progression, including the breast tumor marker PS2,11 were differentially expressed in the microdissected cell populations. This study demonstrates the feasibility of applying LCM and nucleic acid expression array technology to histologically complex clinical specimens for the purposes of molecularly dissecting human neoplasia.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
All human tissue specimens were obtained from the Alvin J. Siteman
Cancer Center Tissue Procurement Core facility using an institutional
review board-approved protocol. A detailed protocol for tissue
preparation and LCM is available at
http://pathbox.wustl.edu/
tisscore/protocols.htm. Embedded
frozen-tissue specimens were cut at 6 µm thickness and immediately
fixed in 70% ethanol. Cut sections were stored at 4°C in 70%
ethanol for up to 72 hours before staining and dissection. We have
previously shown that storage of sections in this manner maintains
reasonable tissue morphology, facilitates transfer of tissue to
transfer film, and provides excellent preservation of cellular RNA
(MAW, manuscript in preparation). Subsequently, tissue sections were
stained as previously described.12
In brief, slides were
sequentially dipped five times in deionized water, 10 times in Mayer’s
hematoxylin solution (Sigma, St. Louis, MO), deionized water, 1x
automation buffer (Biomeda, Foster City, CA), and deionized water.
Slides were then dehydrated for 60 seconds each in 70% ethanol and
95% ethanol, stained for 15 seconds in alcoholic eosin (Sigma), rinsed
with 10 dips in 95% ethanol, and placed for 60 seconds in 95%
ethanol. Finally, slides were further dehydrated by 10 dips in 100%
ethanol, two 60-second washes in 100% ethanol, 10 dips in xylene, and
two incubations of 3 minutes each in xylene. Slides were air-dried for
5 minutes and stored in a dessicator for no more than 6 hours before
dissection. Nonmalignant ductal epithelial cells and foci of carcinoma
in situ were isolated from the slides using the PixCell II
LCM System (Arcturus Engineering, Mountain View,
CA). Between 4300 and 6800 laser pulses (30-µm beam diameter; 30 mW
power) yielded an estimated average of 15,000 cells.
RNA Extraction
For RNA dilution experiments, total cellular RNA was isolated from a single culture flask of MDA-MB361 human mammary tumor cells using the RNeasy RNA isolation system (Qiagen, Valencia, CA) and following manufacturer’s recommendations. RNA was assessed by formaldehyde agarose gel electrophoresis, quantitated by UV absorbance, and diluted to a concentration of 1 µg/µl. For microdissected tissue samples, several LCM caps were pooled into a single tube containing 200 µl of denaturing buffer guanidinium isothiocyanate (GITC) and 1.6 µl of ßME. Total RNA was then extracted using a modified protocol of the Stratagene RNA microisolation kit (Stratagene, La Jolla, CA) as previously described.6 The total RNA obtained from each LCM-dissected tissue was resuspended in 10 µl of RNase-free water. To assess the quality and concentration of the total RNA, 1 µl was directly analyzed on an RNA LabChip (Agilent, Wilmington, DE) following the manufacturer’s instructions.
Internal Controls
Plasmid constructs encoding transcriptional templates for the B. subtillis genes lys, phe, trp, and thr were obtained from the American Type Culture Collection (Manassas, VA). Purified plasmid DNAs were used to generate in vitro, polyadenylated transcripts using the Megascript kit (Ambion, Austin, TX) and following the manufacturer’s protocol. RNA was purified using RNeasy spin columns (Qiagen) and quantitated by UV absorbance. Each of the transcripts was mixed to yield a cocktail of 1 x 106 copies/µl trp, 5 x 106 copies/µl thr, 2 x 107 copies/µl phe, and 1 x 108 copies/µl lys. Before synthesis of first strand cDNA, transcript cocktail was added to the RNA sample at a final concentration of 1 x 106 copies of trp per 10 µg of total RNA. This corresponds approximately to the four synthetic transcripts being present at 1:2000 (lys) to 1:200,000 (trp) endogenous transcripts.
Target Synthesis
A detailed protocol for target synthesis is available at
http://pathbox.wustl.edu/mgacore/protocols.htm. For biotin-labeled
target synthesis starting from 10 µg of cell line total RNA,
reactions were performed using standard protocols supplied by the
manufacturer (Affymetrix) and as previously described.13
For target synthesis starting from 10 ng of total RNA or LCM-extracted
RNA, an initial round of amplification was performed before synthesis
of biotin-labeled cRNA. For the first round of amplification, synthesis
of first- and second-strand cDNA was performed using the standard
protocol provided by the manufacturer (Affymetrix) and as previously
described.13
However, instead of proceeding to use the
double-stranded cDNA in the biotin-labeled in vitro
transcription reaction, the cDNA was resuspended in 8 µl of
RNase-free water and used as a template to transcribe unlabeled
antisense RNA (aRNA) using T7 RNA polymerase and the Megascript kit
(Ambion). The reaction was incubated for 4 hours at 37°C and the
resulting aRNA was purified using RNeasy spin columns (Qiagen). Eluted
aRNA was precipitated by adding 0.1 volume of 7.5 mol/L ammonium
acetate, 0.02 volumes of 5 mg/ml linear acrylamide (Ambion), and 2.5
volumes of 100% ethanol, and resuspended in 10 µl of RNase-free
water. A second round of amplification was initiated by using the aRNA
as template. After annealing aRNA with 0.7 µmol/L random hexamers
(Pharmacia, Piscataway, NJ) for 10 minutes at 70°C, the mixture was
chilled on ice and extended in a 20-µl reaction containing 4 µl of
5x first-strand reaction buffer, 2 µl of 0.1 mol/L dithiothreitol, 1
µl of 10 mmol/L dNTPs, and 1 µl of Superscript II (Life
Technologies, Rockville, MD). After a 1-hour incubation at 42°C, 1
µl of 2 U/ml of RNase H was added, incubated for 20 minutes at
37°C, and inactivated at 95°C for 5 minutes. The resulting
first-strand cDNA was annealed to 100 pmol of high performance liquid
chromotography (HPLC)-purified T7T24 primer
(Genset, La Jolla, CA) for 10 minutes at 70°C. Then, second-strand
cDNA synthesis was performed by adding 90 µl of RNase-free water, 30
µl of 5x second-strand reaction buffer, 3 µl of 10 mmol/L dNTPs,
10 U DNA ligase, 40 U DNA polymerase, and 2 U of RNase H. After
incubating the second-strand cDNA reaction for 2 hours at 16°C, 20 U
of T4 DNA polymerase were added, followed by incubation at 16°C for
10 minutes. The second-strand cDNA synthesis was stopped by adding 10
µl of 0.5 mol/L ethylenediaminetetraacetic acid. Double-stranded cDNA
was purified by phenol:chloroform:isoamyl alcohol extraction using
phase-lock-gel (Eppendorf, Westbury, NY), precipitated with 0.5 volumes
of 7.5 mol/L ammonium acetate, 2 µg of glycogen, and 2.5 volumes of
100% ethanol, and resuspended in 22 µl of RNase-free water.
Biotin-Labeled cRNA Transcription and Gene-Chip Hybridization
Biotinylated cRNA target was generated from both amplified and nonamplified cDNAs using the Bioarray high-yield transcription kit (Enzo, New York, NY) following the manufacturer’s protocol. After a 5-hour incubation at 37°C, the final biotin-labeled cRNA product was purified using RNeasy spin columns (Qiagen) and eluted in 40 µl of RNase-free water. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 25 µg of each biotinylated cRNA preparation was fragmented, assessed by gel electrophoresis, and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix Hu6800SubD GeneChip arrays for 16 hours. GeneChips were washed and stained using the instrument’s standard Eukaryotic GE Wash 2' protocol, using antibody-mediated signal amplification.
Data Analysis
The images from the scanned chips were processed using Affymetrix
Microarray Analysis Suite 4.0. In the first set of validation
experiments that used duplicate RNA samples, the image from each
GeneChip was individually scaled such that the average intensity value
for all arrays was adjusted to 1500. Scaled average difference value,
log average ratio, and absolute call data from each GeneChip
were exported to flat text files and used for numerical analysis. For
analysis of clinical specimens, the GeneChip image of the carcinoma
in situ sample was normalized to the corresponding image of
the nonmalignant epithelium sample, across all probe pair sets.
Difference call, fold change, average difference value, and absolute
call data from each of the three specimen pairs analyzed were
exported to flat text files. The complete set of data may be downloaded
from the site: http://pathbox.wustl.edu/mgacore/.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Table 1
provides a summary of results
from this first experiment. The scaling factor is a coefficient used to
multiply signal intensities of each probe set so that the average
signal intensity over the entire chip reaches a common target intensity
(in this case 1500 units). Roughly speaking, it is a measure of total
hybridization signal intensity. Although the total mass of biotinylated
target hybridized to the array was comparable for all three samples,
the hybridization signal (as reflected by the scaling factor) was
approximately twofold lower in samples derived from 10 ng of total RNA
as compared to that from 10 µg. Another means to score the assay
sensitivity is to examine the number of genes scored as detected
("P") versus nondetected ("A") within each sample.
For the 10-µg sample, 685 of 1879 (36%) probe sets were scored
"P" whereas in the two 10-ng samples 538 (29%) and 428 (23%) were
scored "P". Looking at individual transcript controls, high copy
number transcripts (ie, lys) showed comparable levels of
signal intensity that, because of the approximate twofold difference in
scaling factors used, resulted in scaled average difference intensity
values that were approximately twofold higher in amplified RNA
versus nonamplified RNA arrays. Moderately abundant
transcripts (ie, phe) showed very similar scaled average
difference values for all three samples whereas lower abundant
transcripts (ie, thr, trp) began to demonstrate a
much higher degree of variability. Furthermore, using the standard
protocol, ratios of lys, phe, and thr
were maintained at an almost perfect fivefold decrement as would be
expected from their corresponding starting copy number of transcripts.
Samples that experienced an additional round of amplification, however,
showed some skewing of this linearity. The endogenous transcripts for
glyceraldehyde-6-phosphate dehydrogenase and ISGF3A, a general
transcription factor, demonstrated comparable behavior. The transcript
for transferrin receptor, however, was detected with 10 µg of
starting RNA, but could not be detected in either sample of amplified
RNA. The number of discordant "P" and "A" calls is also
summarized in Table 1
. Most importantly, the number of discrepant calls
between the two amplified samples is relatively low (3 to 8%) and
comparable to the variability observed between identical sets of
10-µg RNA samples (MAW, unpublished data). As expected, the highest
discordance rate was between transcripts scored as "P" in the
10-µg sample, but "A" in the amplified RNA samples. In summary,
these data suggest that although detection sensitivity is compromised
to some degree and there is some nonlinearity in comparing amplified
versus nonamplified material, the protocol provides
reproducible results from a single additional round of transcript
amplification when starting with 1000-fold less input RNA. Although we
sought to determine the minimum amount of RNA necessary to achieve a
reasonable hybridization signal, we also suspect that some issues of
sensitivity and linearity can be better preserved when greater than 10
ng of starting material is used.
|
shows representative
LCM procurement of nonmalignant ductal cells. Approximately 5000
30-µm laser firings were used to retrieve 15,000 to 20,000 cells
of each specified type. To determine the quality and quantity of
resulting RNA, a 1-µl aliquot (10%) of each sample was analyzed by
microcapillary gel electrophoresis with fluorescence detection (Figure 1C)
. In most cases, RNA retrieved from LCM tissue was of high quality,
as judged by 2:1 band intensity of 28S and 18S ribosomal bands. Typical
yields ranged from 40 to 80 ng of RNA, or 12 ng of RNA per 1000
30-µm laser pulses. In addition to assessing RNA quality,
quantitation of total LCM RNA allowed for the addition of polyA
transcript standards at defined copy number per total mass of input
RNA. Approximately 50 ng of RNA from patient-matched nonmalignant and
malignant cells were amplified and used to generate labeled targets as
described above.
|
shows relative gene expression
values (fold change) between nonmalignant epithelial cells and
corresponding carcinoma in situ from each of the three
patients. In the one patient sample pair where they were included,
internal control transcripts (lys, phe,
thr) varied by no more than 1.6-fold between the
independently amplified nonmalignant and malignant samples. Endogenous
housekeeping control genes (glyceraldehyde-6-phosphate dehydrogenase,
ß-actin, and ISGF3A) also demonstrated no more than 1.5-fold
differences between a single patient sample pair as well as between all
three patient pairs.
|
lists several genes that demonstrated concordant
changes in expression of >2.5-fold between the nonmalignant and
in situ carcinoma samples in at least two of the three
patients. Fold change values in parentheses represent estimates, as one
of the measured hybridization signals fell below the background level.
Not surprisingly, no one gene showed consistent changes in expression
among all three patient pairs. Given the reasonable analytical and
molecular reproducibility of the assay as demonstrated (Table 1)
and
the consistency of internal controls and housekeeping genes, these
differences likely represent the inherent biological variability that
can be expected when analyzing expression profiles between
patients.4 Despite the small number of patient samples and gene probes used in the present study, two distinct expression profiles were apparent. Lactoferrin, a marker of estrogen stimulation in breast epithelium,15 was up-regulated in ductal carcinoma in situ (DCIS) from patients 1 and 2, but relatively down-regulated, relative to nonmalignant epithelium in patient 3. In contrast, PS2, a well-characterized estrogen-responsive marker of breast tumor progression11 and SIX1, a homeobox protein frequently up-regulated in metastatic breast cancer,16 were only overexpressed in patient 3, and to a lesser extent, patient 1. Two other genes, hevin and oxytocin receptor, were also down-regulated in DCIS versus the normal duct epithelium of patients 1 and 3, but not patient 2. Hevin/SC1, a SPARC-like extracellular matrix-associated glycoprotein important for cell adhesion and migration, is frequently down-regulated in prostate adenocarcinoma as well as several other tumor types.17 The peptide hormone oxytocin can inhibit growth of breast cancer cells and expression of the corresponding oxytocin receptor has been well characterized in both nonmalignant and malignant mammary epithelium.18 Intriguingly, transcription of the oxytocin receptor is also regulated by c-fos,19 a second gene down-regulated in one, but not all three, nonmalignant/malignant specimen pairs.
The functional significance of these and other findings from this data set will require additional studies using a larger cohort of specimens, additional numbers of probe pair sets, and more sophisticated bioinformatics approaches. Of more immediate importance, this study demonstrates how LCM can be easily coupled to high-density oligonucleotide array technology to obtain expression profiles from discrete cell populations in histologically complex clinical specimens. Specifically, we have found that careful preparation and staining of tissue sections, dissection of a large (but not burdensome) number of cells, qualitative and quantitative assessment of isolated cellular RNA, and inclusion of a set of internal transcript controls to monitor assay fidelity can greatly contribute to the success of this approach. Whether the molecular profile of an isolated sample of specific tumor cells or the more comprehensive tumor microenvironment will provide the best clinical diagnostic tool remains to be determined. In any event, the approach described here will be of obvious benefit to more precisely dissect the pathobiology and molecular evolution of disease processes like human breast cancer in complex tissue specimens.
|
Footnotes |
|---|
Supported in part by grant no. 016-99 from the Mary Kay Ash Charitable Foundation.
Accepted for publication March 2, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  T. L. Adair-Kirk, J. J. Atkinson, G. L. Griffin, M. A. Watson, D. G. Kelley, D. DeMello, R. M. Senior, and T. Betsuyaku Distal Airways in Mice Exposed to Cigarette Smoke: Nrf2-Regulated Genes Are Increased in Clara Cells Am. J. Respir. Cell Mol. Biol., October 1, 2008; 39(4): 400 - 411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Wilhelm, J. P. Muyal, J. Best, G. Kwapiszewska, M. M. Stein, W. Seeger, R. M. Bohle, and L. Fink Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments Clin. Chem., June 1, 2006; 52(6): 1161 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. S. Schuetz, M. Bonin, S. E. Clare, K. Nieselt, K. Sotlar, M. Walter, T. Fehm, E. Solomayer, O. Riess, D. Wallwiener, et al. Progression-Specific Genes Identified by Expression Profiling of Matched Ductal Carcinomas In situ and Invasive Breast Tumors, Combining Laser Capture Microdissection and Oligonucleotide Microarray Analysis. Cancer Res., May 15, 2006; 66(10): 5278 - 5286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Lawlor, L. Soucek, L. Brown-Swigart, K. Shchors, C. U. Bialucha, and G. I. Evan Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel Insights into Myc-Mediated Tumorigenesis. Cancer Res., May 1, 2006; 66(9): 4591 - 4601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. King, N. Guo, G. M. Frampton, N. P. Gerry, M. E. Lenburg, and C. L. Rosenberg Reliability and Reproducibility of Gene Expression Measurements Using Amplified RNA from Laser-Microdissected Primary Breast Tissue with Oligonucleotide Arrays J. Mol. Diagn., February 1, 2005; 7(1): 57 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Matsuzaki, M. Canis, C. Vaurs-Barriere, J.L. Pouly, O. Boespflug-Tanguy, F. Penault-Llorca, P. Dechelotte, B. Dastugue, K. Okamura, and G. Mage DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection Mol. Hum. Reprod., October 1, 2004; 10(10): 719 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Schmid, C. D. Cohen, A. Henger, D. Schlondorff, and M. Kretzler Gene expression analysis in renal biopsies Nephrol. Dial. Transplant., June 1, 2004; 19(6): 1347 - 1351. [Full Text] [PDF]
|
|
|
|
 |
|
 P.-P. Zheng, A. M. Sieuwerts, T. M. Luider, M. van der Weiden, P. A.E. Sillevis-Smitt, and J. M. Kros Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature Am. J. Pathol., June 1, 2004; 164(6): 2217 - 2228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Burstein, K. Polyak, J. S. Wong, S. C. Lester, and C. M. Kaelin Ductal Carcinoma in Situ of the Breast N. Engl. J. Med., April 1, 2004; 350(14): 1430 - 1441. [Full Text] [PDF]
|
|
|
|
 |
|
 G. Li, M. Cuilleron, A. Gentil-Perret, M. Cottier, K. Passebosc-Faure, C. Lambert, C. Genin, and J. Tostain Rapid and Sensitive Detection of Messenger RNA Expression for Molecular Differential Diagnosis of Renal Cell Carcinoma Clin. Cancer Res., December 15, 2003; 9(17): 6441 - 6446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S D Jenson, R S Robetorye, S D Bohling, J A Schumacher, J W Morgan, M S Lim, and K S J Elenitoba-Johnson Validation of cDNA microarray gene expression data obtained from linearly amplified RNA Mol. Pathol., December 1, 2003; 56(6): 307 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sanchez-Carbayo, F. Saint, J. J. Lozano, A. Viale, and C. Cordon-Cardo Comparison of Gene Expression Profiles in Laser-Microdissected, Nonembedded, and OCT-Embedded Tumor Samples by Oligonucleotide Microarray Analysis Clin. Chem., December 1, 2003; 49(12): 2096 - 2100. [Full Text] [PDF]
|
|
|
|
|
|
G. J. Miller, H. L. Miller, A. van Bokhoven, J. R. Lambert, P. N. Werahera, O. Schirripa, M. S. Lucia, and S. K. Nordeen Aberrant HOXC Expression Accompanies the Malignant Phenotype in Human Prostate Cancer Res., September 15, 2003; 63(18): 5879 - 5888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Diggle, S. Cruickshank, J. D. Olsburgh, S. Pellegrin, B. Smith, R. E. Banks, P. J. Selby, M. A. Knowles, J. Southgate, and P. Harnden Identification of Genes Up-Regulated in Urothelial Tumors: The 67-kd Laminin Receptor and Tumor-Associated Trypsin Inhibitor Am. J. Pathol., August 1, 2003; 163(2): 493 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-J. Ma, R. Salunga, J. T. Tuggle, J. Gaudet, E. Enright, P. McQuary, T. Payette, M. Pistone, K. Stecker, B. M. Zhang, et al. Gene expression profiles of human breast cancer progression PNAS, May 13, 2003; 100(10): 5974 - 5979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Porter, J. Lahti-Domenici, A. Keshaviah, Y. K. Bae, P. Argani, J. Marks, A. Richardson, A. Cooper, R. Strausberg, G. J. Riggins, et al. Molecular Markers in Ductal Carcinoma in Situ of the Breast Mol. Cancer Res., March 1, 2003; 1(5): 362 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kabbarah, K. Pinto, D. G. Mutch, and P. J. Goodfellow Expression Profiling of Mouse Endometrial Cancers Microdissected from Ethanol-Fixed, Paraffin-Embedded Tissues Am. J. Pathol., March 1, 2003; 162(3): 755 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. S. Stappenbeck, J. C. Mills, and J. I. Gordon Molecular features of adult mouse small intestinal epithelial progenitors PNAS, February 4, 2003; 100(3): 1004 - 1009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Luzzi, M. Mahadevappa, R. Raja, J. A. Warrington, and M. A. Watson Accurate and Reproducible Gene Expression Profiles from Laser Capture Microdissection, Transcript Amplification, and High Density Oligonucleotide Microarray Analysis J. Mol. Diagn., February 1, 2003; 5(1): 9 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Adeyinka, E. Emberley, Y. Niu, L. Snell, L. C. Murphy, H. Sowter, C. C. Wykoff, A. L. Harris, and P. H. Watson Analysis of Gene Expression in Ductal Carcinoma in Situ of the Breast Clin. Cancer Res., December 1, 2002; 8(12): 3788 - 3795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Wulfkuhle, D. C. Sgroi, H. Krutzsch, K. McLean, K. McGarvey, M. Knowlton, S. Chen, H. Shu, A. Sahin, R. Kurek, et al. Proteomics of Human Breast Ductal Carcinoma in Situ Cancer Res., November 15, 2002; 62(22): 6740 - 6749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kretzler, C. D. Cohen, P. Doran, A. Henger, S. Madden, E. F. Grone, P. J. Nelson, D. Schlondorff, and H.-J. Grone Repuncturing the Renal Biopsy: Strategies for Molecular Diagnosis in Nephrology J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1961 - 1972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Leo, S. Y. Hsu, and A. J. W. Hsueh Hormonal Genomics Endocr. Rev., June 1, 2002; 23(3): 369 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ernst, M. Hergenhahn, M. Kenzelmann, C. D. Cohen, M. Bonrouhi, A. Weninger, R. Klaren, E. F. Grone, M. Wiesel, C. Gudemann, et al. Decrease and Gain of Gene Expression Are Equally Discriminatory Markers for Prostate Carcinoma : A Gene Expression Analysis on Total and Microdissected Prostate Tissue Am. J. Pathol., June 1, 2002; 160(6): 2169 - 2180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Jeffrey, M. J. Fero, A.-L. Borresen-Dale, and D. Botstein Expression Array Technology in the Diagnosis and Treatment of Breast Cancer Mol. Interv., April 1, 2002; 2(2): 101 - 109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Gutmann, N. M. Hedrick, J. Li, R. Nagarajan, A. Perry, and M. A. Watson Comparative Gene Expression Profile Analysis of Neurofibromatosis 1-associated and Sporadic Pilocytic Astrocytomas Cancer Res., April 1, 2002; 62(7): 2085 - 2091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Fink, S. Kohlhoff, M. M. Stein, J. Hanze, N. Weissmann, F. Rose, E. Akkayagil, D. Manz, F. Grimminger, W. Seeger, et al. cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue Am. J. Pathol., January 1, 2002; 160(1): 81 - 90. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
