Improved Sensitivity for FFPE Microarray Analysis: ArrayGrade FFPE RNA Isolation

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1 Improved Sensitivity for FFPE Microarray Analysis: ArrayGrade FFPE RNA Isolation Improved RNA Isolation from FFPE Blocks and Slides for Greater Microarray Sensitivity By Savita Prabhakar, George Quellhorst, Yexun (Bill) Wang, and Xiao Zeng SABiosciences 6951 Executive Way Frederick, MD (301) Phone +1 (301) Fax ABSTRACT This paper describes a new method for isolating RNA from formalin-fixed paraffinembedded (FFPE) blocks and slides that improves gene expression profiling sensitivity and the number of positive calls in both high-density genome-wide and low-density pathway-focused microarray techniques. The key to enhanced sensitivity is a more effective reversal of the chemical cross-links from RNA caused by the fixation process. The ArrayGrade FFPE RNA Isolation Kit more readily isolates RNA from fixed tissues and provides a better template for microarray labeling protocols than other commercial kits. Scientific data presented in this white paper demonstrates that this method isolates RNA from FFPE samples with greater yield, purity, and real-time PCR sensitivity. Most importantly, the RNA isolated by this method also provides percent positive calls that are more comparable to fresh tissue samples using both the Agilent DNA microarrays and SABiosciences' Oligo GEArrays. The ArrayGrade FFPE RNA isolation method now brings microarray-based gene expression profiling to researchers with access to archived tissue samples. GEArray is a registered trademark of SABiosciences. ArrayGrade, ReactionReady, RT 2 Real-Time and XpressRef are all trademarks of SABiosciences. SYBR Green is a registered trademark of Molecular Probes, Inc. icycler is a registered trademark of Bio-Rad Laboratories.

2 Introduction: Archived Formalin Fixed, Paraffin-Embedded (FFPE) tissue samples potentially represent an invaluable source for gene expression analysis data. FFPE samples are the most readily available material from human disease studies for which pathology and clinical outcomes are known and well documented. Analyzing gene expression patterns or profiles in these archived tissues should facilitate retrospective studies correlating gene expression patterns with the relevant disease states. Such profiles may serve as additional biomarkers for disease classification, staging and other diagnostic or prognostic purposes. RNA extracted from FFPE blocks is often too cross-linked to effectively serve as a substrate for the enzyme reactions involved in gene expression analysis (1, 2). Therefore, some have concluded (3, 4) that traditional microarray platforms are not suitable for gene expression analysis of the RNA extracted from FFPE blocks. In one notable study, Okubo s group demonstrated that mono-methylol modification of fixed RNA is the key factor that makes the nucleic acid a poor template for reverse transcription (1). Their recommended protocol changes, however, do not improve the performance of FFPE RNA in a microarray experiment (5), suggesting that they do not completely reverse the cross-linking between RNA and protein (1). Currently available commercial FFPE RNA isolation kits either fail to include a cross-linking reversal step or perform cross-linking reversals in sub-optimal buffers resulting in overly fragmented RNA that is not suitable microarray labeling protocols. In fact, the experimental results provided to support these kits do not offer any microarray data and instead focus solely on real-time PCR performance. This white paper describes SABiosciences method for RNA extraction from FFPE blocks and slides that yields better results on microarrays than other commercial or published methods. The ArrayGrade FFPE RNA Isolation method includes a heating step in a proprietary buffer system that more effectively reverses formalin-induced cross-links. This protocol enhancement allows the RNA to be not only more readily purified, but also a better template for the enzymatic reactions that convert RNA into labeled target for microarray hybridization. The ArrayGrade FFPE RNA isolation method is also compatible with multiple microarray platforms providing researchers with more ways to analyze their samples and find more candidate genes for biomarker discovery, screening, and validation. Benefits of ArrayGrade FFPE RNA Isolation: Improved RNA Isolation: More effectively reverses cross-links than other methods. Greater Real-Time PCR Sensitivity: Lowers real-time PCR threshold cycle values. Greater Microarray Sensitivity: Increases present positive calls on microarrays. 2 ArrayGrade FFPE RNA Isolation

3 Improved RNA Isolation: Quantity and Quality: The ArrayGrade FFPE RNA Isolation Method provides a higher yield of RNA from mouse heart, kidney, and liver FFPE blocks when compared to other commercial RNA isolation kits (Table 1). The ArrayGrade method also yields RNA of greater purity from protein contamination (as evidenced by the A260:A280 ratios) and from organic compounds (as evidenced by the A260:A230 ratios). The ArrayGrade method isolates RNA of sufficient purity for microarray analysis with A260:A280 ratios greater than 2.0 and A260:A230 ratios greater than 1.7 more consistently than the other commercial kits used in this study µ 1.0 cm x 0.5 cm each µ 0.4 cm x 0.8 cm each µ 0.4 cm x 0.5 cm each Kidney Yield (µg) A260:A280 A260:A230 ArrayGrade 32.7* Kit A 11.8* Kit Q Kit I Liver Yield (µg) A260:A280 A260:A230 ArrayGrade 26.2* Kit A 11.5* Kit Q ND Kit I Heart Yield (µg) A260:A280 A260:A230 ArrayGrade 7.0* Kit A ND Kit I Kit Q Table 1: The ArrayGrade FFPE RNA Isolation Method Isolates More RNA Of A Greater Purity Than Other Commercial Kits. RNA was isolated from replicate sets of sections cut from FFPE blocks of mouse kidney, liver, or heart organs using the ArrayGrade method an either two or three other commercial kits ( A, Q, or I ). The yields and purities, expressed as the A260:A280 and A260:A230 ratios, of the RNA samples are listed. Absorption ratios with a check mark ( ) pass while those without fail the quality control necessary for microarray analysis. Yields with an asterisk (*) are sufficient for standard protocols on both low-density and high-density microarrays, while those without are only sufficient for lowdensity microarray applications. (ND = Not Determined) The size of the RNA isolated by the ArrayGrade FFPE RNA Isolation Method is more compatible with microarray analysis than RNA isolated by other methods. In their attempts to reverse FFPE RNA cross-links, two commercially available RNA isolation kits tested in this study ( I and Q ) use a heating step in a buffer system not well suited to this purpose. The resulting RNA is very small averaging around only 200 base pairs in length (Figure 1) and is thus overly fragmented for microarray analyses, which require as much as 1 kbp of RNA length. In contrast, another RNA isolation kit ( A ) lacks a heating step, resulting in RNA that is rather large indicating that a great deal of crosslinking remains. Extensive cross-linking inhibits the reverse transcriptases used in enzymatic microarray labeling protocols. The ArrayGrade FFPE RNA Isolation method includes a moderate heating step in a unique buffer system optimized for the reversal of FFPE cross-links. The resulting RNA size provides a middle ground between no heat treatment and harsh heating procedures. ArrayGrade FFPE RNA Isolation 3

4 Kit Q Kit I ArrayGrade Kit A Figure 1: The ArrayGrade FFPE RNA Isolation Method Provides Better RNA Integrity. The size distribution of RNA isolated from sections of a mouse kidney FFPE block using the ArrayGrade Total RNA Isolation Kit as well as Kits A, I, and Q were characterized with an RNA NanoChip 6000 on an Agilent BioAnalyzer. Improved RNA Performance: Greater Real-Time PCR Sensitivity RNA isolated by the ArrayGrade method yields lower real-time PCR threshold cycle (C t ) values indicating greater limits of detection and sensitivity for housekeeping genes than the other commercial methods (Table 2). In fact, the ArrayGrade C t values are closer to the values seen from fresh tissue RNA when compared to other methods. The apparent RNA size distribution differences and greater real-time PCR sensitivity together indicate a greater degree of cross-linking reversal. We then tested whether the ArrayGrade method yields more intact and readily available RNA for optimal microarray detection. ACTB GAPD C t C t Minus RT C t C t Minus RT Fresh N/A N/A ArrayGrade N/A N/A Kit Q Kit I Kit A N/A Table 2: ArrayGrade FFPE RNA Isolation Provides Greater Real-Time PCR Sensitivity. RNA samples isolated from sections of a mouse liver FFPE block by the four different methods or from fresh mouse liver were converted to template using the ReactionReady First Strand cdna Synthesis Kit (C- 01). PCR was performed on the Bio-Rad icycler using the RT 2 Real-Time SYBR / Fluorescein PCR Master Mix (PA-011) and RT 2 Real-Time qpcr Primer Sets for Mouse ACTB and GAPDH (PPM02945A and PPM02946A). The threshold cycle (C t ) values of both genes in all RNA samples are displayed as well as the difference in the C t values ( C t ) between fresh and FFPE tissue. To determine the extent of genomic DNA contamination, the corresponding no reverse transcription control C t values are also listed. Check marked ( ) C t values less than 35 pass while those greater than 35 fail the necessary quality control for accurate real-time PCR analysis. 4 ArrayGrade FFPE RNA Isolation

High-Density Genome-Wide Agilent DNA Microarrays: Improved Sensitivity and More Positive Calls A more vigorous test for FFPE RNA quality is DNA microarray analysis.

5 High-Density Genome-Wide Agilent DNA Microarrays: Improved Sensitivity and More Positive Calls A more vigorous test for FFPE RNA quality is DNA microarray analysis. As pointed out by Okubo s group (1), the poly-a tails of FFPE mrna are heavily modified with monomethylol groups and do not anneal well with oligo-dt primers. Most target labeling protocols for microarrays employ an IVT protocol that depends on the annealing of an oligo-dt-t7 primer. Therefore, the success of the FFPE cross-linking reversal is directly observable as improved microarray sensitivity. Many researchers prefer to analyze the expression profile of the entire genome to glean as much information as possible from precious FFPE samples. RNA isolated from mouse kidney FFPE blocks by the ArrayGrade method yields percent present calls and signal intensities on the high-density Agilent microarrays that are more comparable to fresh tissue RNA than FFPE RNA extracted by the other commercial methods (Figure 2 and Table 3). Fresh ArrayGrade Kit Q Kit I Figure 2: ArrayGrade FFPE RNA Isolation Yields More Positive Calls on Agilent microarrays than Other Commercial Methods. RNA samples isolated from sections of a mouse kidney FFPE block, with either ArrayGrade or two other commercial methods, and RNA from fresh tissue were characterized on the Mouse Oligo Microarray Kit (V2) from Agilent according to the manufacturer s instructions. Representative images from the same sections of each microarray are displayed with a few spots circled to emphasize the relative intensity level of these genes observed in each RNA sample. RNA Isolation Method Significantly Above Background Well Above Background Correlation with Fresh Tissue Fresh N/A ArrayGrade y = 0.724x R 2 = Kit Q y = 0.583x R 2 = Kit I y = 0.376x R 2 = Table 3: The ArrayGrade FFPE RNA Isolation Method Provides Genome-Wide Expression Profiles More Comparable with Fresh Tissue than Other Commercial Methods. The percent positive calls from the arrays in Figure 2 were calculated and expressed as either Significantly Above Background, meaning the number of mean signal intensities above background based on a two-sided t- test, or Well Above Background, meaning the number of background-corrected signal intensities greater than 2.6 standard deviations about the average background. The fresh tissue signal intensities were graphed against the intensities from each FFPE RNA isolation method in separate scatter plots (data not shown). The curve fits to a straight line were determined and also listed in the table highlighting how close the slopes and the correlation coefficients are to the ideal of 1.0. ArrayGrade FFPE RNA Isolation 5

The expression levels of the detectable genes in the ArrayGrade FFPE RNA provide a better correlation to those levels observed with fresh tissue than other commercial kits, based on linear fits to

6 The expression levels of the detectable genes in the ArrayGrade FFPE RNA provide a better correlation to those levels observed with fresh tissue than other commercial kits, based on linear fits to scatter plots comparing the data (Table 3). These results demonstrate that the ArrayGrade protocol makes RNA from FFPE samples more available as a substrate for the enzymatic reactions required for high-density microarray analysis. Pathway-Focused Low-Density Oligo GEArrays : Improved Sensitivity and More Positive Calls Other researchers prefer to simplify the analysis of their FFPE samples by focusing on genes related to a specific disease state, or biological pathway, or otherwise related to their specific area of research. RNA isolated from FFPE blocks of mouse kidney (Figure 2), heart, and liver (as summarized in Table 3) by these methods also yields percent present calls more comparable to fresh tissue RNA on the pathway-focused Oligo GEArray microarrays than FFPE RNA extracted by other commercial methods. Fresh ArrayGrade Kit Q Kit A Kit I Figure 3: ArrayGrade FFPE RNA Isolation Provides More Positive Calls on the Oligo GEArray than Other Commercial Methods. Equal amounts of RNA (3 µg) isolated either from fresh mouse kidney or FFPE mouse kidney sections using the ArrayGrade method and three other commercial kits were used to generate crna with the TrueLabeling-AMP 2.0 Kit (GA-030) in an overnight reaction. Labeled crna (3.0 µg) was hybridized overnight to identical copies of the Mouse Tumor Metastasis Oligo GEArray in the HybPlate Format (EMM-028). The raw microarray images are displayed demonstrating more visible spots from the RNA isolated by the ArrayGrade method than the other commercial methods. Percent Positive Calls RNA Isolation Method Kidney Heart Liver Fresh ArrayGrade Kit Q ND Kit A 16.0 ND 22.7 Kit I Table 4: ArrayGrade FFPE RNA Isolation Provides More Positive Calls on the Oligo GEArray than Other Commercial Methods. Summarized are the results of the experiment in Figure 2 as well similar comparisons between the RNA isolation methods using fresh tissue and FFPE block sections of mouse heart and liver. The percent present call for each sample is displayed as determined by the GEArray Expression Analysis Suite. 6 ArrayGrade FFPE RNA Isolation

7 Conclusions: The value of characterizing FFPE samples using emerging molecular technologies has been well recognized (6). Unlike approaches that modify microarray labeling protocols after RNA extraction (5, 7, 8), the ArrayGrade method follows Okubo s lead (1) and focuses on the basic problem of FFPE RNA quality. This white paper demonstrates that the method extracts RNA with a greater yield and purity that is more compatible with microarray-based characterization of gene expression. The intermediate size of the RNA and the greater real-time PCR sensitivity indicates a level of cross-linking reversal that does not overly fragment the RNA. Most importantly, the RNA isolated from FFPE blocks and slides by the ArrayGrade method visibly increases the present call for highdensity genome-wide as well as low-density pathway-focused DNA microarrays. References: 1. Masuda, N., Ohnishi, T., Kawamoto, S., Monden, M. and Okubo, K. (1999) Nucleic Acids Res, 27, Godfrey, T.E., Kim, S.-H., Chavira, M., Ruff, D.W., Warren, R.S., Gray, J.W. and Jensen, R.H. (2000) J Mol Diagn, 2, Cronin, M., Pho, M., Dutta, D., Stephans, J.C., Shak, S., Kiefer, M.C., Esteban, J.M. and Baker, J.B. (2004) Am J Pathol, 164, Bibikova, M., Talantov, D., Chudin, E., Yeakley, J.M., Chen, J., Doucet, D., Wickham, E., Atkins, D., Barker, D., Chee, M. et al. (2004) Am J Pathol, 165, Karsten, S.L., Van Deerlin, V.M., Sabatti, C., Gill, L.H. and Geschwind, D.H. (2002) Nucleic Acids Res, 30, E4. 6. Paik, S., Kim, C.Y., Song, Y.K. and Kim, W.S. (2005) Nat Clin Pract Oncol, 2, Lehmann, U., Bock, O., Glockner, S. and Kreipe, H. (2000) Pathobiology, 68, Specht, K., Richter, T., Muller, U., Walch, A., Werner, M. and Hofler, H. (2001) Am J Pathol, 158, Perform more sensitive microarray-based gene expression profiling and analysis with the ArrayGrade FFPE RNA Isolation Kit. Use your archived clinical and pathology samples to find candidate genes for biomarker discovery, screening, and validation. Ordering Guide: Product: ArrayGrade FFPE RNA Isolation Kit Catalog Number: GA-023 ArrayGrade FFPE RNA Isolation 7

Rapid Method for the Purification of Total RNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Samples

Application Note 17 RNA Sample Preparation Rapid Method for the Purification of Total RNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Samples M. Melmogy 1, V. Misic 1, B. Lam, PhD 1, C. Dobbin,