1Department of Anatomy & Physiology, Kansas State University, Manhattan, Kansas; and 2NuGEN Technologies, Inc., San Carlos, California
Submitted 25 May 2004 ; accepted in final form 17 December 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
gene expression microarray analysis; microdissection; nucleic acid amplification techniques
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse kidney (MK) total RNA (Ambion, Austin, TX) and mouse universal reference (MUR) total RNA (Stratagene, La Jolla, CA) were chosen as sources for RNA to minimize biological variation and interexperimental differences. Targets were prepared using four different methods: 1) one round of amplification (OneRA), 2) two rounds of amplification (TwoRA), 3) Ribo-SPIA linear amplification with the Ovation biotin system (RS), and 4) Ribo-SPIA linear amplification with the picogram Ribo-SPIA prototype system (pRS).
One round of amplification. Starting with 10 µg of total RNA, cRNA targets were prepared using OneRA [standard protocol, version VII; Affymetrix (see https://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf)] (Fig. 1). Briefly, RNA was transcribed into cDNA using reverse transcriptase with a T7 primer that contains a promoter for DNA-dependent RNA polymerase (3, 21). After RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA (dscDNA) was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was performed in the presence of T7 RNA polymerase and a biotinylated nucleotide analog-ribonucleotide mix for cRNA amplification and biotin labeling. The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip expression arrays.
|
Ribo-SPIA linear amplification. Ribo-SPIA-based RNA amplifications and target preparations were performed according to the manufacturer's instructions (picogram Ribo-SPIA prototype system, pRS and Ovation Biotin System, RS; see http://www.nugeninc.com/technology/index.shtml) (Fig. 2). Briefly, RNA was reverse transcribed into cDNA using reverse transcriptase with a DNA/RNA chimeric primer that is part DNA and part RNA. RNA was degraded by heating, and fragments served as primers for second-strand synthesis, yielding a dscDNA with an RNA/DNA heteroduplex at one end. The RNA portion of the heteroduplex portion of the dscDNA was digested using RNase H added to the reaction mixture together with a DNA polymerase and a second chimeric cDNA/cRNA primer (amplification primer). Amplification was continued using primer extension, strand displacement, and degradation of the RNA portion of the primer extension product hybridized to the target to reveal part of the priming site for subsequent primer hybridization and extension by strand displacement DNA synthesis. Accumulated cDNA amplification products were fragmented and labeled to generate biotinylated cDNA targets. cDNA targets were prepared using RS starting with 100, 30, 10, or 3 ng of total RNA or pRS starting from 10, 3, or 0.3 ng of total RNA.
|
Quantities of total RNA, cRNA, and cDNA were determined using absorbance spectrophotometry (ND-1000 spectrophotometer; NanoDrop Technologies, Wilmington, DE). For total RNA and cRNA, the conventional conversion, 1 OD260 = 40 ng/µl, was used. For cDNA, the manufacturer's (NuGEN Technologies) recommended conversion, 1 OD260 = 33 ng/µl, was used. The quality of total RNA (eukaryotic total RNA nano assay) and cRNA and cDNA (mRNA smear nano assay) was determined using microfluidic electrophoresis (Bioanalyzer; Agilent Technologies, Palo Alto, CA).
Microarray Analysis
Hybridizations of cRNA and cDNA targets were performed according to the manufacturer's recommended procedures on high-density oligonucleotide gene chips (Affymetrix Mouse Genome 430 2.0 GeneChip arrays; see http://www.affymetrix.com/support/technical/datasheets/mogarrays_datasheet.pdf). A total of 39 target preparations were performed, and each preparation was analyzed using one GeneChip array. Data were scaled to a target intensity of 500 (GCOS software; Affymetrix). Normalization quality controls, including scaling factors, average intensities, present calls, background intensities, noise, and raw Q values all were within acceptable limits (Table 1). Hybridization controls, BioB, BioC, BioD, and CreX, were called present on all chips and yielded the expected increases in intensities. Analyses of target populations were supported by GeneSpring (Silicon Genetics, Redwood City, CA), Excel (Microsoft, CA), and Origin 6.0 (OriginLab, Northampton, MA). Microarray data were deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (accession nos. GSE1435 and GSE2019).
|
Signal correlation. Signal intensities without regard to present, absent, or marginal calls were correlated, and linear correlation coefficients (r) were determined (Origin, version 6.0). Correlation r values within a set of triplicates were reported as averages ± SD of the three possible correlations. Correlation r values between two sets of triplicates were based on averaged intensities and reported as a simple r value.
Sensitivity.
Sensitivity was evaluated from the estimated fold change necessary for significance (Foldsig). Foldsig was obtained according to Foldsig = [Avg + 2.776 x (2/3 x SD2)]/Avg, where Avg and SD are the average and standard deviation of the signal intensity of genes called present in all replicates, and 2.776 is the t value at P = 0.05, assuming 4 df.
Differential gene expression. The signal intensity ratios of genes detected in all targets prepared from 10 µg and 10 ng MK and MUR RNA using OneRA, TwoRA, RS, and pRS were calculated from the averages of triplicates. Ratios were correlated between amplification systems, and correlation coefficients were obtained.
Verification of Microarray Data
A small number of gene targets amplified from MUR RNA and detected using microarrays were verified by performing RT-PCR (Table 2). Targets were chosen on the basis of gene array data obtained from 10 ng of MUR RNA amplified using either TwoRA or RS. For each method, 13 targets were selected on the basis of six criteria established to ensure fair selection between these two methods. 1) Targets must be called present in three replicates of the considered amplification method (TwoRA or RS). 2) Targets must be called absent in three replicates of the other amplification method. 3) Hybridization intensities of the three replicates of the considered amplification method must be >100. 4) Targets of the considered amplification method must be annotated by Affymetrix as full-length mRNA and not as expressed sequence tag. 5) Hybridization intensities between sets of chosen targets must be comparable. 6) Fold differences between called present using one amplification method and absent using the other method must be comparable between sets of chosen targets. Average intensities and calls for the 26 selected genes are shown in Table 2, not only for TwoRA and RS but also for OneRA and pRS.
|
Statistics
Gene array experiments were performed in triplicate. RT-PCR experiments were performed at least in duplicate, mostly in quadruplicate. Data, including quality control values, are expressed as averages ± SD. The significance of continuous data was determined using one-way ANOVA with a Bonferroni post hoc test. Significance was assumed at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Samples of total RNA ranging from 0.3 ng to 10 µg were used as starting materials. Quantities were verified spectrophotometrically. RNA qualities were evaluated by performing microfluidic electrophoresis (Fig. 3). MUR RNA was of slightly higher quality than MK RNA as evidenced by the larger ratio between the 28S and 18S peak. cRNA targets were prepared using OneRA or TwoRA starting with 10 µg or 10 ng RNA, respectively. Alternatively, cDNA targets were prepared using RS starting with 3, 10, 30, or 100 ng of RNA and using pRS starting with 0.3, 3, and 10 ng of RNA. Figure 4 summarizes cRNA and cDNA target yields obtained after amplification. Yields for cRNA and cDNA targets were significantly different, but each was sufficient for preparation of the hybridization cocktail (Affymetrix). The size distributions of amplified products and of biotinylated and fragmented targets are shown in Fig. 5.
|
|
|
|
|
|
|
|
A sizable number of targets (n = 13,995; 13,539 + 456, based on MUR RNA) were amplified from 10 ng of RNA using both TwoRA and RS (Fig. 7). An additional 5,929 (2,546 + 3,383) targets were TwoRA specific because they were amplified using TwoRA and not RS, and an additional 3,093 (845 + 2,248) targets were RS specific because they were amplified using RS and not TwoRA. TwoRA- and RS-specific targets had similar distributions of hybridization intensities and of fold differences between hybridization intensities that were called present and called absent. TwoRA- and RS-specific target populations were sampled according to a set of criteria (see MATERIALS AND METHODS) that ensured fair comparison between these two systems. All 13 TwoRA-specific and all 13 RS-specific targets could be verified in MUR RNA (Table 1), a finding consistent with equal fidelity of TwoRA and RS. Of the 26 selected targets, 15 were called present using OneRA and 24 were called present using pRS. Quantitative comparisons other than between TwoRA and RS are not warranted, given that unbiased selection was ensured only for TwoRA and RS. Taken together, these data demonstrate equal fidelity of TwoRA and RS and suggest that each amplification system amplifies a unique set of targets in addition to the overlapping target sets.
Reproducibility between Replicates
Call concordances and correlation coefficients within triplicate samples were calculated to evaluate reproducibility between replicates (Table 4; the best examples are shown in Figs. 9, AD, and 10). Between replicates, call concordances were similar for targets prepared using OneRA or TwoRA and somewhat lower for RS and pRS (92.2, 91.8, 89.3, and 88.1%, respectively, for target prepared from 10 µg and 10 ng of MUR RNA). Signal correlations were similar for targets prepared using all amplification systems.
|
|
Direct comparison. Call concordances were obtained from comparisons of samples prepared from 10 µg of RNA using OneRA and from 10 ng using TwoRA, RS, or pRS (Table 5). Call concordances between OneRA and TwoRA were higher than for any other comparison. Observations were similar for MK and MUR RNA. Signal intensities were correlated between amplification systems (Fig. 9, EH). Correlation coefficients between OneRA and TwoRA and between RS and pRS were better, most likely because of the greater similarities between these amplification systems. Low call concordances and signal correlations between T7- and Ribo-SPIA-based systems suggest that the two amplification methods may introduce different biases or that cDNA and cRNA perform differently on GeneChip arrays. Indeed, cDNA/DNA hybridizations may be more reliable than cRNA/DNA hybridization because of the lesser complexity of cDNA/DNA interactions (17, 18, 24), which may affect both present vs. absent calls and signal intensities.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RS and pRS generated significantly smaller amounts of cDNA than TwoRA generated cRNA (Fig. 4), although all systems produced sufficient amounts of targets, given that 15 µg of cRNA and only 2 µg of cDNA are needed for array hybridization. Yields of cDNA targets prepared using RS and pRS were independent of the amount of starting material, which is similar to the pattern observed with PCR.
Probe sets for 18S rRNA were low in signal intensity and inconsistently called present in targets prepared using OneRA. However, 18S rRNA was consistently called present, with high signal intensities in target preparations prepared using TwoRA, RS, and pRS (Fig. 6). This observation may indicate that both T7- and Ribo-SPIA-based amplification systems prime at internal polyA sites. Alternatively, it is conceivable that polyadenylated forms 18S rRNA may be present as recently observed in yeast (10). Thus it is possible that the higher sensitivity afforded by RNA amplification enables the detection of small amounts of polyadenylated rRNA on the arrays. The majority of amplified transcripts generated using RS and pRS were up to 1,000 nt in length (Fig. 5). A similar length distribution was found for TwoRA. In contrast, the majority of amplified transcripts generated using OneRA were up to 2,500 nt in length. These differences are not likely to be important on Affymetrix arrays, in which >98% of the probe sets are located <600 nt from the 3' end.
3' Bias was further determined for three genes with probe sets that varied in their distances from the 3' end of the mRNA (Table 3). 3'/M and 3'/5' ratios for GAPDH are best suited to predict fidelity of expression profiling given that >98% of probe sets on Affymetrix Mouse Genome 430 2.0 GeneChips are specific for sequences within 600 nt from the 3' end. The observation that 3'-M and 3'-5' ratios for GAPDH were close to unity is consistent with the view that TwoRA, RS, and pRS amplify targets with high fidelity. The observation that 3'-5' ratios for the transferrin receptor, which are based on probe sets located 528 and 2,225 nt from the 3' end, could be obtained only for OneRA is consistent with the finding that OneRA yielded longer transcripts than TwoRA, RS, or pRS.
The RNA starting material for MK and MUR differed notably in quality (Fig. 3). Consistently, significant differences between MK and MUR RNA were found for probe sets that are located 972 and 1,665 nt from the 3' end (3'-5' ratio for -actin; Fig. 11). However, no significant differences were found for ratios of probe sets located 387 and 770 nt from the 3' end (3'-M ratio for GAPDH; Fig. 11). Given that >98% of probes are located within 600 nt of the 3' end, the difference in RNA quality was not expected to have a major effect on the gene array data. Consistently, analyses of GeneChip array data from both RNA gave similar results.
|
Reproducibility between replicates was based on call concordance and estimated fold changes necessary for significance (Table 4 and Fig. 8). Reproducibility for targets that were prepared from 10 ng of RNA using TwoRA was higher than for targets prepared from 10 ng of RNA using RS or pRS. Fold changes necessary for significance, however, were well below 2 for all amplification systems. A fold change of 2 is sometimes used as an arbitrary lower limit for significance.
As expected, comparisons of targets populations prepared using different amplification methods yielded poorer call concordances and signal intensity correlations than comparisons of targets populations prepared using the same method (9, 14). This difference illustrates the presence of system-specific biases.
Call concordances were higher between the two T7-based systems, OneRA and TwoRA, than between T7- and Ribo-SPIA-based systems (Table 5), most likely because of the greater similarity of the methods and the similar number of present calls. Although RS and pRS are similar methods, they differed greatly in the number of present calls, which resulted in poorer call concordance. Signal correlation coefficients within the two T7-based (OneRA and TwoRA) or within the two Ribo-SPIA-based methods (RS and pRS) were significantly higher than any comparison between these methods (Table 5 and Fig. 9, EH). The observation that absent vs. present calls between OneRA and TwoRA or between OneRA and pRS were significantly larger than present vs. absent calls is consistent with amplification of rare messages using TwoRA and pRS. Amplification is likely to raise signal intensities of rare genes above the noise level (6).
Differential gene expression was computed in an attempt to cancel system-dependent biases. Nevertheless, correlation between ratios of targets prepared using T7-based systems, TwoRA and OneRA, was considerably better than that between ratios of T7- and Ribo-SPIA-based methods (Fig. 9, GI). This observation underscores the presence of system-specific biases.
All nanogram amplification methods, TwoRA, RS, and pRS, yielded sufficient material for gene array work, although TwoRA yielded quantitatively more cRNA than RS or pRS yielded cDNA. The RS and pRS target preparation methods produced results comparable to those observed using more traditional T7-based methods and will enable studies of smaller RNA samples because the required input level is lower and the time and effort required for amplification are lower. pRS reproducibly amplified the highest number of targets and was found to be suitable for amplification of total RNA from amounts as low as 0.3 ng. Reproducibility and sensitivity of TwoRA relative to OneRA were higher than those of RS or pRS. All amplification systems, OneRA, TwoRA, RS, and pRS, amplified large overlapping sets of targets. Target preparations using RS and pRS were faster and produced cDNA, which is more stable than cRNA and thus can be banked for additional studies. The presence of system-specific biases prompts the recommendation that changes in amplification methodology within a study be avoided. Indeed, in the anticipation of future refined studies on nanogram amounts RNA, investigators may want to choose a nanogram amplification system for a pilot study even if microgram amounts of RNA are available.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
DISCLOSURES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* R. Singh, R. J. Maganti, and S. V. Jabba contributed equally to this work.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Deng G, Dafforn A, Wang M, Chen P, Purohit R, Wang S, Pillarisetty S, Iglehart D, Koritala S, Lato S, Herrler M, Heath J, Stanchfield J, and Kurn N. A new method for amplification and labeling of RNA from small clinical samples for use with the Affymetrix GeneChip platform (Abstract 1507). Poster presented at the 53rd annual meeting of the American Society of Human Genetics, Los Angeles, CA, 2003.
3. Eberwine J. Amplification of mRNA populations using aRNA generated from immobilized, oligo(dT)-T7 primed cDNA. Biotechniques 20: 584591, 1996.[ISI][Medline]
4. Feldman AL, Costouros NG, Wang E, Qian M, Marincola FM, Alexander HR, and Libutti SK. Advantages of mRNA amplification for microarray analysis. Biotechniques 33: 906914, 2002.[ISI][Medline]
5. Heath J, Brooks A, Richfield E, Thiruchelvam M, Cory-Slechta D, Wang M, Chen P, Dafforn A, Deng G, Iglehart D, Koritala S, Lato S, Pillarisetty S, Purohit R, Herrler M, Stanchfield J, and Kurn N. Comparative gene expression analysis of microdissected brain tissues in a mouse model of idiopathic Parkinson's disease using a novel RNA amplification system (Abstract 1553). Poster presented at the 53rd annual meeting of the American Society of Human Genetics, Los Angeles, CA, 2003.
6. Hu L, Wang J, Baggerly K, Wang H, Fuller GN, Hamilton SR, Coombes KR, and Zhang W. Obtaining reliable information from minute amounts of RNA using cDNA microarrays. BMC Genomics 3: 16, 2002.[CrossRef][Medline]
7. Iscove NN, Barbara M, Gu M, Gibson M, Modi C, and Winegarden N. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat Biotechnol 20: 940943, 2002.[CrossRef][ISI][Medline]
8. Karsten SL, Van Deerlin VMD, Sabatti C, Gill LH, and Geschwind DH. An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis. Nucleic Acids Res 30: E4, 2002.[CrossRef][Medline]
9. Kenzelmann M, Klären R, Hergenhahn M, Bonrouhi M, Gröne HJ, Schmid W, and Schütz G. High-accuracy amplification of nanogram total RNA amounts for gene profiling. Genomics 83: 550558, 2004.[CrossRef][ISI][Medline]
10. Kuai L, Fang F, Butler JS, and Sherman F. Polyadenylation of rRNA in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 101: 85818586, 2004.
11. Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, and Brown EL. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol 14: 16751680, 1996.[CrossRef][ISI][Medline]
12. Luzzi V, Mahadevappa M, Raja R, Warrington JA, and Watson MA. Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J Mol Diagn 5: 914, 2003.
13. Makrigiorgos GM, Chakrabarti S, Zhang Y, Kaur M, and Price BD. A PCR-based amplification method retaining the quantitative difference between two complex genomes. Nat Biotechnol 20: 936939, 2002.[CrossRef][ISI][Medline]
14. McClintick JN, Jerome RE, Nicholson CR, Crabb DW, and Edenberg HJ. Reproducibility of oligonucleotide arrays using small samples. BMC Genomics 4: 4, 2003.[CrossRef][Medline]
15. Pabon C, Modrusan Z, Ruvolo MV, Coleman IM, Daniel S, Yue H, and Arnold LJ Jr. Optimized T7 amplification system for microarray analysis. Biotechniques 31: 874879, 2001.[ISI][Medline]
16. Puskas LG, Zvara A, Hackler L Jr, and van Hummelen P. RNA amplification results in reproducible microarray data with slight ratio bias. Biotechniques 32: 13301340, 2002.[ISI][Medline]
17. Rosenow C, Saxena RM, Durst M, and Gingeras TR. Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches. Nucleic Acids Res 29: e112, 2001.
18. Schwille P, Oehlenschläger F, and Walter NG. Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis. Biochemistry 35: 1018210193, 1996.[CrossRef][ISI][Medline]
19. Spiess AN, Mueller N, and Ivell R. Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations. BMC Genomics 4: 44, 2003.[CrossRef][Medline]
20. Stears RL, Getts RC, and Gullans SR. A novel, sensitive detection system for high-density microarrays using dendrimer technology. Physiol Genomics 3: 9399, 2000.
21. Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, and Eberwine JH. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci USA 87: 16631667, 1990.
22. Wang E, Miller LD, Ohnmacht GA, Liu ET, and Marincola FM. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18: 457459, 2000.[CrossRef][ISI][Medline]
23. Wangemann P, Itza EM, Albrecht B, Wu T, Jabba SV, Maganti RJ, Lee JH, Everett LA, Wall SM, Royaux IE, Green ED, and Marcus DC. Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model. BMC Med 2: 30, 2004.[CrossRef][Medline]
24. Wu P, Nakano S, and Sugimoto N. Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation. Eur J Biochem 269: 28212830, 2002.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |