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From the Divisions of Population Science
* and Medical Science,
Fox Chase Cancer Center, Philadelphia, Pennsylvania; and Arcturus Bioscience,
Sunnyvale, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A new RNA processing system (Paradise Reagent System; Arcturus Bioscience), which allows robust isolation and amplification of RNA from FFPE samples, has been developed. Two primary advantages of this methodology over other published protocols for gene expression profiling of RNA from FFPE tissue9, 10 are its adaptation to the analysis of severely limited amounts of homogeneous cells (as little as 5 ng of total RNA) as acquired by laser capture microdissection (LCM) and the combined use of linear amplification to generate a sufficient amount of RNA for microarray analyses. When compared with RNA amplified by PCR, the RNA amplified linearly using T7 RNA polymerase and oligo(dT) primers exhibits both a greater extent of amplification and a stronger correlation with the expression profile of nonamplified samples.11, 12 Although reports have begun to emerge on the successful use of the Paradise Reagent System for gene expression profiling of FFPE tissue by quantitative real-time PCR,13 an in-depth comparison of microarray data generated from FFPE tissue with that obtained from frozen specimens has not been performed and is essential to validate this promising methodology.
The goal of the present study is to assess the feasibility of obtaining reliable microarray data from FFPE samples collected from resection specimens obtained during routine surgical procedures. This evaluation was accomplished by comparing the gene expression profiles of FFPE samples with those of matched frozen tissue samples obtained from the same patient, the latter representing an optimal source of material for microarray analysis. A further objective is to establish predictive markers, such as the integrity and size of the total RNA obtained from FFPE tissue, as indicators of the anticipated quality of downstream microarray data.
This report demonstrates that it is possible to obtain credible microarray data from FFPE tissue, even when a small amount of RNA, acquired by LCM, is used. A set of practical recommendations for evaluating RNA integrity and quality control, which can guide the implementation of the developed technology in any laboratory setting, has been established. Because formalin fixation-paraffin embedding is the universal protocol for routine pathological diagnosis, the proposed procedure represents an invaluable tool with which to correlate the genomic characteristics of a sample with the wealth of morphological and clinical information available for the same specimen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the gene expression profile using oligonucleotide arrays, five pairs of frozen and FFPE specimens were obtained from the archives of the Department of Pathology. Four of the formalin-fixed surgical specimens (subjects 26, 27, 62, and 68) were fixed within 45 to 90 minutes upon arrival from the operating room and placed in the tissue processor after 7 to 28 hours of formalin fixation, as per the routine departmental protocol. The sample from subject 201 was fixed in formalin immediately upon delivery from the operating room and processed within 24 hours of fixation. All frozen specimens were immediately placed in liquid nitrogen upon arrival in the Department of Pathology and subsequently stored at –80°C.
Tissue Sectioning and LCM
Frozen sections (5 µm thickness) were cut at –20°C and immediately transferred to a microslide box kept on dry ice and stored at –80°C. FFPE sections were cut (5 µm thickness), floated in an RNase-free water bath, and transferred to glass slides. These slides were placed under the hood to dry for 1 hour and subsequently stored in a microslide box at –80°C. A new blade was used for each frozen and FFPE sample.
To obtain a homogeneous population of epithelial cells for analysis, LCM was performed using tissue sections from both FFPE and frozen samples. Immediately before LCM, the slide box was taken from the freezer and placed on dry ice. FFPE samples were deparaffinized, stained using Histogene Solution (Arcturus Bioscience), and dehydrated; 5000 malignant cells were laser capture microdissected from colon tumors using the automated AutoPix System (Arcturus Bioscience). The process described above was also followed for frozen tissues, except for the deparaffinization step. Representative images of the LCM procedure are presented in Figure 1
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For oligonucleotide microarray analyses, total RNA (11 µl) from five matched pairs of FFPE and frozen microdissected tissue was subjected to two rounds of linear RNA amplification based on T7 polymerase in vitro transcription. The first round of amplification was performed using the Paradise Reagent System and the RiboAmp OA kit (Arcturus Bioscience) for FFPE and matched frozen samples, respectively. The second round of amplification was performed subsequently using the Paradise Reagent System for both FFPE and matched frozen samples. A total of 10 (five FFPE and five frozen) labeled antisense (aRNA) probes for microarray hybridization were generated during the second round of amplification in the presence of 5-[3-aminoallyl] uridine 5'-triphosphate (UTP) after indirect incorporation of Cy3 fluorescent dye (Amersham Biosciences Corp., Piscataway, NJ). One hundred nanograms of Human Universal Reference total RNA (BD Biosciences, Palo Alto, CA) was amplified in parallel following the same protocol used for RNA derived from frozen samples and labeled with Cy5 fluorescent dye (Amersham Biosciences Corp.). The concentration of the labeled aRNA (picomoles per microliter) was determined using an ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
Oligonucleotide Microarrays
Microarray hybridizations were performed using a custom-designed 22,000 oligonucleotide (60-mer) array commercially available from Agilent Technologies. The microarray also contains 314 genes that serve as negative controls. This array was designed to contain probes preferentially located in the 3' end of the transcripts. Cy3-labeled aRNA (75 pmol for FFPE and 55 pmol for frozen samples) was mixed with Cy5-labeled reference aRNA (55 pmol) and hybridized onto the microarray in an Agilent G2545A Hybridization Oven using the In Situ Hybridization kit Plus (Agilent Technologies), as described by the manufacturer. The resulting 10 microarrays were scanned using an Agilent DNA Microarray Scanner (model G2565BA).
Data Quantification and Statistical Analysis
Spot and local background intensities of the acquired microarray images were quantified using Feature Extraction Software v.7.5 (Agilent Technologies). The log (base 10) expression ratios of the dye-normalized intensities were calculated by the software. The expression ratio of each gene was determined and expressed as the dye-normalized intensities of the reference sample over the corresponding value for the test (frozen or FFPE) sample. The software also reports the P value of the ratios as a measure of the confidence that the gene is differentially expressed in the comparison between reference (Cy5 channel) and test samples (Cy3 channel) (Agilent Feature Extraction Software v.7.5 User Guide).
The subsequent analyses included two steps: 1) determination of the expressed genes and 2) assessment of the reproducibility and differences between the expression profiles of FFPE and frozen specimens. The intensity of each spot (both the Cy3 and Cy5 channels) was first filtered for good quality, based on the default parameters provided by the Feature Extraction Software. Briefly, a spot was defined as good if it was not flagged as a saturation and nonuniformity outlier and was positive [significantly above background (Agilent Feature Extraction Software v.7.5 User Guide)] in a single channel. A gene was defined as expressed ("E") if it was represented by a good spot in both the Cy3 and Cy5 channels, and the P value of the ratio was below 0.05. Otherwise, a gene was considered nonexpressed ("N"). The percentage of noncontrol genes (N = 21,939) expressed was calculated as a measure of chip performance. In addition, the percentage of expressed genes detected in negative controls (N = 314) was used to identify potential false-positive signals in each microarray.
The reproducibility between matched frozen and FFPE samples was assessed based on 1) the Pearson’s correlation coefficients and Spearman’s rank order correlation coefficients using the expression ratios of the expressed genes, in matched frozen and FFPE samples and 2) the concordance scores of expressed and nonexpressed genes in matched frozen and FFPE samples.
Reverse Transcription (RT)-PCR
To evaluate the length of the aRNA, which reflects the extent of degradation of the input total RNA, primers specific for human ß-actin (HBAC), keratin-20 (KRT20), and N-acetyltransferase 1 (NAT-1) were designed to target different regions of the mRNA sequence. The primer sequences and corresponding details are found in Table 1
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) for 30 seconds, 72°C for 30 seconds, and a final cycle of 72°C for 10 minutes. Products were resolved using standard 2% agarose gel electrophoresis and visualized by ethidium bromide staining under UV light. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 18S and 28S ribosomal RNA peaks are observed in all frozen and FFPE samples, even though the profile of RNA recovered from FFPE samples is more heterogeneous. Both the broadening of ribosomal RNA peaks and the presence of fragmentation products between the 18S ribosomal subunit and the control marker (first peak in the electropherogram) indicate that RNA quality is affected by tissue fixation. In all instances, the quality of RNA extracted from cells isolated by LCM (Figure 2
, large panels) was identical to that of scraped specimens (Figure 2
, small panels). No significant differences were observed with respect to RNA quality when the tissues were processed immediately (0 hours) or after 2 hours. However, a change in the baseline between the marker and the 18S ribosomal peak was observed in frozen samples processed after 2 hours, indicating partial RNA degradation.
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shows the RNA quality of five paired samples obtained from the Fox Chase Cancer Center archives that were subjected to LCM and gene expression analyses. Similar to Figure 2
, RNA extracted from epithelial cells of frozen tissue showed 28S and 18S ribosomal RNA peaks, indicative of good-quality total RNA. In contrast, the profiles of RNA isolated from FFPE tissue appeared more heterogeneous than those presented in Figure 2
. The electropherograms included a single peak (samples 26 and 27) or a plateau (samples 62 and 68), except for sample 201-FFPE, where ribosomal RNA peaks were present.
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. However, the length of the aRNA, as determined by agarose gel electrophoresis, was slightly smaller for FFPE samples compared with frozen-derived aRNAs (maximum length 500 and 400 nucleotides, respectively). The length of the aRNA generated from sample 201-FFPE, which was the only FFPE RNA preserving ribosomal RNA peaks, was comparable to that of aRNA derived from the matched frozen specimen.
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. The presence or absence of an amplicon is a qualitative measure of the length of the cDNA template. cDNA from all frozen and most FFPE samples was amplified successfully using these primer sets. No product was generated when FFPE samples 26 and 27 were subjected to amplification with primers KRT20_3 and NAT-1.
Microarray Data Analysis
The percentage of false positives and expressed genes (as defined in Materials and Methods) along with the dye-normalized mean signal intensities in both channels are summarized in Table 2
. The false-positive rate (ie, the percentage of negative control genes detected as expressed) is insignificantly low across all hybridizations. The percentage of genes expressed was very similar in matched frozen and FFPE samples—differing by approximately 5% in samples 27, 62, and 201 and by 13 and 12% in samples 26 and 68, respectively.
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. Among the five paired specimens, sample 201 presented the highest score (42.5%) of expressed genes and the lowest percentage (34.5%) of nonexpressed genes of all samples evaluated.
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. The default P value cutoff used to define expressed genes is 0.05 (as described in Materials and Methods). In addition, the impact of different numbers of expressed genes on the similarity of gene expression profiles was explored by varying the P value cutoff that was used to define the expressedgenes. By making the P value cutoff more rigorous (from 0.05 to 0.01 and 0.001), fewer expressed genes and slightly higher correlation coefficients were observed. The impact on the correlation coefficients is less for those samples with better input RNA quality (1 to 2% for samples 201 and 62) than those samples with poor input RNA quality (4 to 5% for samples 26 and 27). This observation confirmed that the similarity of gene expression profiles between FFPE and frozen samples is less sensitive to the selection of genes for those samples with better RNA quality. The scatter plots in Figure 5
illustrate the overall distribution of expression ratios using expressed genes (P < 0.05) in matched FFPE and frozen samples. Samples with lower correlation coefficients (26, 27, and 68) had genes that were differentially expressed more than 10-fold in matched specimens. The highest Pearson’s correlation coefficient (r = 0.96) was observed for the "EE" in sample 201.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the establishment of distinct expression profiles for specific cell types, it is no longer optimal to evaluate transcript levels in mixed cell populations.1, 14 LCM ensures accurate data interpretation by restricting cell collection to only those cells of interest and circumventing contamination from other tissue components.
One of the primary limitations of microarray analysis is the large amount of labeled input RNA (5 to 10 µg) required for hybridization.15
Thus, the minute quantities of RNA extracted from LCM material are insufficient for global gene expression profiling, dictating the need for RNA amplification. Moreover, reliable strategies to maintain a high correlation between the expression profile of amplified and nonamplified samples, such as linear amplification based on T7 RNA polymerase and oligo(dT) primers,11
should be used. The Paradise Reagent System (Arcturus Bioscience) had been designed to address both the requirement for a reliable amplification protocol and the compromised quality of FFPE specimens. Extraction of RNA from 5000 colonic epithelial cells, isolated by LCM, yielded as little as 5 ng of total RNA, an amount sufficient to perform microarray analysis after two rounds of linear RNA amplification using the Paradise Reagent System. Although others have evaluated the gene expression profile of RNA from FFPE samples using different approaches,6, 8, 9, 10, 16
the characteristics that the input RNA must exhibit to support these analyses have not been reported. The present study represents a direct comparison of the quality of total RNA extracted from FFPE and frozen colon tissues after LCM and scraping. The resulting data demonstrate that the integrity of RNA extracted from both frozen and FFPE tissue is not affected by LCM (Figure 2)
, suggesting that one can reliably use scraped tissue to determine the RNA quality of the sample before LCM, a laborious and expensive procedure. Surprisingly, a 2-hour delay in processing of the surgical specimen in the Surgical Pathology Laboratory did not significantly affect the quality of the RNA extracted from either frozen or fixed specimens (Figure 2
, bottom panels). As illustrated in Figures 2
and 3
(right panels), the quality of RNA obtained from FFPE tissue is quite variable. Although ribosomal RNA peaks are preserved in some cases, fluorescence is detected by the Bioanalyzer over a longer period of time. This profile is observed especially if the tissue is processed within 2 hours after collection. The highest quality of RNA was derived from FFPE sample 201. This sample, which was subjected to immediate formalin fixation, presented the highest correlation values with the paired frozen specimen. This observation validates the importance of using the Bioanalyzer as a quality control tool. In contrast, ribosomal RNA peaks were absent in the remaining FFPE samples. Extensive variability in the length of the RNA produced a plateau (FFPE samples 62 and 68), whereas the prevalence of short RNA fragments, indicative of degradation, yielded a sharp peak within the first seconds of the electropherogram run (FFPE samples 26 and 27). Based on these criteria, the FFPE samples can be ranked with respect to RNA quality (highest to lowest): 201, 62, 68, 26, and 27.
The demonstrated success in obtaining high-quality RNA from sample 201-FFPE as well as the samples in Figure 2
can be explained by the fact that this surgical specimen was fixed in formalin immediately (or within a 2-hour interval) after collection in the operating room. An additional five samples collected in a similar manner also showed preservation of the ribosomal subunit peaks (data not shown). Although sample 201 was stored for the shortest time, no correlation was observed between the length of archival storage (1 to 5 years) and RNA quality. For example, sample 62, which was stored for 5 years, showed better RNA quality than samples stored for 4 years (samples 26, 27, and 68). Thus, autolysis resulting from a delay in formalin fixation of the surgical specimen, rather than storage time, seems to be the primary factor affecting the quality of extracted RNA. Comprehensive studies designed to establish the optimal conditions to preserve the RNA quality of archived tissues are necessary but will require several years for completion and will not facilitate the use of samples already archived. In this respect, the present study provides guidelines for the use of archived samples already available.
Data from this study indicate that the length of the resulting aRNA is directly related to the method for preserving the specimen (frozen or FFPE). The maximum aRNA length observed was 500 and 400 nucleotides for frozen and FFPE-derived aRNAs, respectively (Figure 4A)
. Because of the high quality of total RNA obtained from sample 201-FFPE, the length of its derived aRNA is similar to that observed in frozen samples. Consistent with other reports,6, 7
the average length of amplified RNA from FFPE specimens is slightly smaller than that obtained from frozen samples. A complementary study to evaluate the length of the aRNA before microarray hybridization was performed using RT-PCR. This reaction assessed the presence or absence of specific portions of the transcripts of HBAC, KRT20, and NAT-1 in both frozen and FFPE-derived RNA. Three of five FFPE samples and all frozen samples were suitable for amplification of fragments up to 214 nucleotides in length and up to 653 nucleotides upstream to the poly-A tail (Figure 4B)
. The fragment amplified using the KRT20_3 primers requires a longer template for successful annealing (Table 1)
and, therefore, is less likely to be detected in low-quality RNAs (such as 26- and 27-FFPE). On the other hand, failure of the NAT-1 fragment to amplify in samples 26- and 27-FFPE is probably due to the fact that this gene is weakly expressed in many tissues, including colon (gene expression data available at Cancer Genome Anatomical Project (http://cgap.nci.nih.gov/Genes/GeneInfo?ORG = Hs&CID = 200738); and its low levels of mRNA might be difficult to detect in RNAs of compromised quality. It should be noted that the HBAC gene, which was amplified successfully in all samples, is a highly expressed housekeeping gene and should not be used exclusively as a marker of RNA quality. Likewise, the same primer sets were not informative when used in quantitative real-time PCR (data not shown).
The results from the present study, using an oligonucleotide array representing approximately 22,000 genes, indicate that the microarray performance obtained from laser microdissected FFPE samples is comparable to that derived from matched frozen samples. Furthermore, the similarities are enhanced when ribosomal RNA peaks can be detected in FFPE samples (Figure 3)
. This result is in agreement with Bibikova et al,10
who reported a high correlation coefficient (R2 = 0.69) when comparing the level of expression of approximately 500 genes in FFPE and fresh-frozen samples. However, microdissection was not used, and higher amounts of input RNA (50 ng) were used. In contrast, other investigators have reported that FFPE tissues are not a reliable substrate for cDNA synthesis and labeling based on the gene expression profile of 95 relatively abundant human genes in matched FFPE and frozen specimens.9
Although a slight loss of sensitivity in signal intensities was observed in the present study when FFPE samples were evaluated (Table 2)
, a high correlation coefficient and significant concordance between expressed genes in both FFPE and frozen samples were obtained (Tables 3
and 4)
. The performance of each sample in the microarray hybridization correlated directly with the quality of the input RNA, as assessed by the Bioanalyzer, RT-PCR, and agarose gel electrophoresis. The concordance among the expressed genes in FFPE and frozen tissue, as indicated by the fraction of expressed genes called "EE," reflects the quality of the total RNA derived from FFPE tissue ranked from highest to lowest: samples 201, 62, 68, 26, and 27. Likewise, a gradual decrease in the fraction of "EE" (0.43, 0.31, 0.27, 0.22, and 0.16, respectively) was observed (Table 3)
. In addition, the high quality of RNA derived from sample 201-FFPE was reflected by the highest correlation coefficient between frozen and FFPE expression ratios (r = 0.96) and the highest fraction of "EE" compared with the fraction of nonexpressed genes ("NN"), using the P value of the expression ratio <0.05. In contrast, sample 27-FFPE exhibited the lowest correlation coefficient (r = 0.80) and the highest fraction of "NN" (Tables 3
and 4)
. Very similar results were observed when different cutoff values were considered for identifying expressed genes (Table 4)
and also when Spearman’s Rho correlation was used (data not shown), confirming the precision of the analysis.
This study demonstrates that the transcript profile of tissue samples that have been archived in laboratories worldwide can now be evaluated for the first time using the Paradise Reagent System. Subsequent correlation of these genomic data with medical history, histopathological findings, and clinical outcome is anticipated to greatly facilitate the development of highly efficacious interventions for a number of diseases.
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Acknowledgments |
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Footnotes |
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Supported by grant CA-06927 from the National Cancer Institute and by an appropriation from the Commonwealth of Pennsylvania.
R.A.C. and S.I.M. are joint first authors.
The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
Accepted for publication August 30, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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500 bases) than that derived from FFPE tissue (