Effects of Globin mRNA Reduction Methods on Gene Expression Profiles from Whole Blood
2006; Elsevier BV; Volume: 8; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2006.060021
ISSN1943-7811
AutoresJinny Liu, Elizabeth Walter, David A. Stenger, Dzung Thach,
Tópico(s)Genomics and Chromatin Dynamics
ResumoExcessive globin mRNA in whole blood RNA decreases transcript detection sensitivity and increases signal variation on microarrays. Hence, methods based on peptide nucleic acid inhibitory oligos and biotinylated DNA capture oligos have been developed to reduce globin mRNA. However, there is limited information about the effects of these two methods on gene expression profiles. Thus, we systematically compared the facility and effects of the two globin reduction methods on profile measurements from Jurkat cell line RNA with or without spiked globin mRNA and human blood RNA isolated using PAXgene collection tubes. We showed that the methods were efficient at increasing the sensitivity of transcript detection without loss of specificity, but neither method could recover a profile equivalent to that of an identical RNA sample without globin mRNA excesses. The capture oligo method had slightly better transcript detection sensitivity for cell line RNA, lowered signal variation for PAXgene RNA, and more similar profiles to controls than the inhibitory method. However, the capture method required larger amounts of initial high-quality RNA to yield sufficient cRNA amounts, and its procedures were more complex and time consuming than the inhibitory method. These results inform the selection of methods suitable for multicenter surveillance of gene expression profiles. Excessive globin mRNA in whole blood RNA decreases transcript detection sensitivity and increases signal variation on microarrays. Hence, methods based on peptide nucleic acid inhibitory oligos and biotinylated DNA capture oligos have been developed to reduce globin mRNA. However, there is limited information about the effects of these two methods on gene expression profiles. Thus, we systematically compared the facility and effects of the two globin reduction methods on profile measurements from Jurkat cell line RNA with or without spiked globin mRNA and human blood RNA isolated using PAXgene collection tubes. We showed that the methods were efficient at increasing the sensitivity of transcript detection without loss of specificity, but neither method could recover a profile equivalent to that of an identical RNA sample without globin mRNA excesses. The capture oligo method had slightly better transcript detection sensitivity for cell line RNA, lowered signal variation for PAXgene RNA, and more similar profiles to controls than the inhibitory method. However, the capture method required larger amounts of initial high-quality RNA to yield sufficient cRNA amounts, and its procedures were more complex and time consuming than the inhibitory method. These results inform the selection of methods suitable for multicenter surveillance of gene expression profiles. Gene expression profile studies of human blood samples in the context of epidemiological surveillance of immunological signatures are confronted by numerous challenges. Previously, we found that increases in hemoglobin from whole blood PAXgene samples of healthy and ill patients contributes to decreased sensitivity of detection of transcripts on Affymetrix microarrays,1Thach DC Agan BK Olsen C Diao J Lin B Gomez J Jesse M Jenkins M Rowley R Hanson E Tibbetts C Stenger DA Walter E Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes.Genes Immun. 2005; 6: 588-595Crossref PubMed Scopus (24) Google Scholar and others have shown that spiking increasing amounts of globin mRNA into total RNA from cell lines results in decreasing detection of transcripts (Affymetrix technical note: Globin Reduction Protocol: A Method for Processing Whole Blood RNA Samples for Improved Array Results. Santa Clara, CA). To curtail this problem, two methods for globin mRNA reduction have been commercially developed. The method from Affymetrix uses the specific binding of peptide nucleic acid (PNA) oligos to the 3′ end of globin mRNA to inhibit reverse transcription during cDNA synthesis.2Debey S Zander T Brors B Popov A Eils R Schultze JL A highly standardized, robust, and cost-effective method for genome-wide transcriptome analysis of peripheral blood applicable to large-scale clinical trials.Genomics. 2006; 87: 653-664Crossref PubMed Scopus (62) Google Scholar The method from Ambion, removes globin mRNA from total RNA via hybridization with biotinylated DNA oligos that specifically capture globin mRNA followed by binding with streptavidin magnetic beads.Both methods seem to offer particular pros and cons depending on the intended application. The established target preparation process normally involves RNA isolation, concentration, cDNA synthesis, and in vitro transcription to generate amplified cRNA.1Thach DC Agan BK Olsen C Diao J Lin B Gomez J Jesse M Jenkins M Rowley R Hanson E Tibbetts C Stenger DA Walter E Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes.Genes Immun. 2005; 6: 588-595Crossref PubMed Scopus (24) Google Scholar,3Thach DC Lin B Walter E Kruzelock R Rowley RK Tibbetts C Stenger DA Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.J Immunol Methods. 2003; 283: 269-279Crossref PubMed Scopus (78) Google Scholar Theoretically, the PNA-based process seems to fit better into the established target preparation process because it is performed in the same tube as the cDNA synthesis reaction, whereas the DNA-based method is a subprotocol that is inserted after total RNA concentration and before cDNA synthesis, therefore taking more time. However, the stability of PNA in the long term is unknown and the method requires taking measures to prevent PNA aggregation and precipitation. Accordingly, the manufacturer recommends that a control RNA sample with spiked-in globin mRNA be run with each batch to ensure that the globin reduction process occurred. In contrast, DNA oligomeric chemistry is more established. Both methods have been shown by their manufacturers to increase detection of transcripts and reduce biological variation between samples. However, it is not known how these methods would transform a gene expression profile, how they compare in practicality and outcomes, or to what extent either method can generate a profile similar to that of samples without high levels of globin mRNA.Therefore, we systematically compared the facility and effects of the two globin reduction methods on gene expression profile measurements from Jurkat cell line RNA with or without spiked globin mRNA and from whole blood RNA isolated from PAXgene tubes. Our results will enable decisions on which method is suitable for incorporation into large-scale multicenter surveillance of gene expression profiles.Materials and MethodsSample CollectionWith approval of the institutional review board at Lackland Air Force Base (San Antonio, TX) and after informed consent, ∼25 ml of blood, filling 10 PAXgene tubes (Preanalytix, Hombrechtikon, Switzerland), were drawn from one healthy volunteer. Blood was drawn into tubes by standard protocol (Preanalytix product circular: PAXgene Blood RNA Tube. Hombrechtikon, Switzerland). All tubes were maintained at room temperature for 2 hours, frozen at −20°C, and shipped on dry ice to the Naval Research Laboratory in Washington, DC, for processing. Tubes were stored at −80°C before RNA isolation.Sample ProcessingBlood collection and RNA isolation was performed using the PAXgene blood RNA system (Qiagen, Valencia, CA), which consists of an evacuated tube for blood collection and a kit for isolation of total RNA from whole blood.4Rainen L Oelmueller U Jurgensen S Wyrich R Ballas C Schram J Herdman C Bankaitis-Davis D Nicholls N Trollinger D Tryon V Stabilization of mRNA expression in whole blood samples.Clin Chem. 2002; 48: 1883-1890PubMed Google Scholar All of the Jurkat cell RNA with or without spiked-in globin mRNA were purchased from Affymetrix (Santa Clara, CA). The isolated RNA underwent further cleanup and concentration followed by globin reduction or no reduction control procedures and was amplified, labeled, and interrogated on the HG-U133 plus 2.0 GeneChip microarrays (Affymetrix).Total RNA Isolation from BloodFrozen PAXgene tubes were thawed at room temperature for 2 hours followed by total RNA isolation as described in the PAXgene blood kit handbook (Preanalytix product insert: PAXgene Blood RNA Kit Handbook. Hombrechtikon, Switzerland) but modified by increasing proteinase K from 40 to 80 μl (>600 mAU/ml) per sample, extending the 55°C incubation time from 10 to 30 minutes, and passing through a QIAshredder spin column (Qiagen). The QIAshredder column shears genomic DNA, thus further facilitating tight pellet formation. The optional on-column DNase digestion was not performed. Purified total RNA was stored at −80°C.1Thach DC Agan BK Olsen C Diao J Lin B Gomez J Jesse M Jenkins M Rowley R Hanson E Tibbetts C Stenger DA Walter E Surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes.Genes Immun. 2005; 6: 588-595Crossref PubMed Scopus (24) Google Scholar,3Thach DC Lin B Walter E Kruzelock R Rowley RK Tibbetts C Stenger DA Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.J Immunol Methods. 2003; 283: 269-279Crossref PubMed Scopus (78) Google ScholarTotal RNA Cleanup and ConcentrationFor more complete removal of DNA from purified PAXgene RNA, replicate RNA samples were pooled followed by in-solution DNase treatment using the DNA-free kit (Ambion, Austin, TX) but without the addition of DNase inactivation reagent because DNase and other contaminants will be removed in the subsequent step. After DNase treatment, RNA was further purified and concentrated using the RNAeasy MinElute Cleanup kit (Qiagen). Subsequently, 1 μl from each sample was run on the Bioanalyzer 2100 (Agilent, Palo Alto, CA) for assessment of RNA quality, whereas the NanoDrop spectrophotometer (ND-1000) was used for quantitation. Usage of the bioanalyzer was analogous to capillary gel electrophoresis. This resulted in electropherograms displaying florescent intensity versus time, which correlates with the amount of RNA versus the size of RNA, respectively.Globin Reduction and Target PreparationThe effects of globin mRNA were alleviated by using the Globinclear (Ambion) or the GeneChip Globin Reduction (Affymetrix) kit, but with modifications. For the Globinclear procedure, biotinylated globin-capture DNA oligos were added to 5 μg of total RNA and globin mRNA were removed by streptavidin magnetic beads. Then the remaining globin-reduced total RNA was purified using magnetic beads and eluted in 30 μl of water. One μl of RNA was used for bioanalyzer measurement and the remaining RNA was concentrated to 8 μl using a Speed Vac (Thermo Electron Corporation, Waltham, MA) at room temperature. For the GeneChip globin reduction procedure, 5 μg of total RNA in 9 μl of BR5 from the RNAeasy MinElute Cleanup eluant was used for cDNA synthesis, whereas the column that came with the globin reduction kit was not used. PNA oligos for inhibition of reverse transcription of globin mRNA were purchased from Applied Biosystems, Foster City, CA. Control mRNA (Affymetrix) at known concentrations was spiked into the samples. These controls were from the lys, phe, thr, and dap genes of the prokaryote Bacillus subtilis and were used because such targets would be absent in eukaryotic samples. All subsequent steps were as described in the GeneChip Expression Analysis Technical Manual, version 701021 Rev. 3.Database IntegrationLaboratory data contained information about the processing of samples from blood in PAXgene tubes to cRNA target preparation, as well as bioanalyzer and NanoDrop measurements. Electropherograms were analyzed by the Biosizing software expert software B.02.01 (Agilent) to output 28S/18S intensity ratios and RNA integrity number (RIN) quality control metrics, whereas the NanoDrop outputs RNA quantity and 260/280 ratios. Report files summarizing the quality of target detection for an array were generated by GeneChip operating software version 1.2 (Affymetrix). JMP (SAS, Cary, NC) was used to join these various data tables together into a metadata table. For gene expression data, signal values were calculated using the Microarray Suite 5.0 algorithm with and without scaling to test it effects on various downstream analytical methods.Statistical AnalysisStatistical quality control and relations among metadata variables and microarray results were analyzed in JMP. Analysis of variance and multidimensional scaling of gene expression data were performed in Arraytools 3.3.0 beta 3a developed by Richard Simon and Amy Lam (http://linus.nci.nih.gov/BRB-ArrayTools.html). Heat maps and dendrograms were graphed using dChip 1.3.5Li C Hung Wong W Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application.Genome Biol. 2001; 2 (RESEARCH0032.1-0032.11)Google Scholar,6Li C Wong WH Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection.Proc Natl Acad Sci USA. 2001; 98: 31-36Crossref PubMed Scopus (2701) Google ScholarResultsQuality of RNA, Globin Reduction, and Target PreparationThree types of RNA samples were used to study the effects of globin reduction methods on gene expression profiles: Jurkat RNA isolated from Jurkat cell line (J), Jurkat RNA with globin mRNA spiked-in (JG), or PAXgene RNA from whole blood (B). Three techniques were tested: using biotinylated globin capture DNA oligos (D), using globin reverse transcription inhibitory PNA oligos (P), or no globin reduction treatment as technical control (C) (Figure 1).The same lot of Jurkat and Jurkat-globin RNA was used throughout. After globin reduction with capture oligos, 77 to 95% of Jurkat and 80 to 91% of Jurkat-globin RNA was recovered (Table 1, second versus first row); the lowered recovery trend for the latter likely reflected globin mRNA removal. This lowered amount of RNA for cDNA synthesis probably accounted for the subsequently lowered cRNA yields for the capture oligo technique (Table 1, third row). Electropherograms of cRNA from Jurkat-globin RNA undergoing globin reduction via capture or inhibitory oligo technique resulted in smaller globin peaks compared with no reduction control (Figure 2A). They also became similar to traces of cRNA from Jurkat RNA without any spiked-in globin mRNA, which were similar regardless of technique (Figure 2A, only JC is shown). These results suggested that for high-quality RNA from a cell line the globin reduction techniques were mostly efficient and did not change the cRNA population size distribution.Table 1Quality Metrics of RNA, Globin Reduction, Target Preparation, and Microarray Results among RNA Type and Technical TreatmentJurkat RNATreatmentCapture DNA oligoInhibition PNA oligoControlStarting material (μg)444Yields after treatment3.56 ± 0.41*STDEV.44Adjusted cRNA yield71.13 ± 5.4196.4 ± 30.66113.47 ± 40.77Scale factors4.50 ± 1.383.98 ± 0.624.42 ± 0.52Background64.21 ± 12.4668.47 ± 11.3060.91 ± 3.713′/5′ GAPDH1.06 ± 0.041.05 ± 0.031.09 ± 0.073′/5′ Actin1.33 ± 0.151.23 ± 0.061.31 ± 0.03Present calls (%)46.8 ± 1.1845.5 ± 0.6244.8 ± 1.65Jurkat-globin RNAPAXgene blood RNACapture DNA oligoInhibition PNA oligoControlCapture DNA oligoInhibition PNA oligoControl4445553.43 ± 0.24443.71 ± 0.325558.33 ± 2.91107.93 ± 29.99124.27 ± 30.9625.87 ± 3.9130.61 ± 17.0541.18 ± 7.765.13 ± 1.065.10 ± 0.505.41 ± 0.897.78 ± 1.827.40 ± 1.7110.68 ± 0.7156.06 ± 3.1870.90 ± 5.8686.6 ± 4.2257.59 ± 3.1961.27 ± 5.5854.27 ± 5.171.06 ± 0.071.09 ± 0.101.14 ± 0.021.70 ± 0.113.59 ± 1.862.25 ± 0.111.25 ± 0.011.17 ± 0.051.05 ± 0.032.55 ± 0.305.94 ± 3.743.16 ± 0.2641.53 ± 0.8337.4 ± 0.732.37 ± 1.5639.33 ± 1.3838.53 ± 2.3932.77 ± 1.39Data in each column are from triplicates.* STDEV. Open table in a new tab Figure 2The globin reduction techniques efficiently reduce globin overabundance from cRNA derived from cell line or PAXgene RNA. Electropherograms of cRNA derived from Jurkat, Jurkat-globin, or PAXgene blood RNA that underwent various technical treatments. A: Traces of cRNA derived from Jurkat-globin RNA treated with DNA capture oligo (JGD), with PNA inhibitory oligo (JGP), or no treatment control (JGC), and from Jurkat RNA with no treatment control (JC). B: Traces of cRNA derived from PAXgene RNA treated with DNA capture oligo (BD), PNA inhibitory oligo (BP), or no treatment control (BC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)PAXgene RNA was pooled from tubes collected from one volunteer. The RIN value of RNA in the eluant of the MinElute column was 8.2 to 9.3. After globin reduction with capture oligos, 75 to 80% of PAXgene RNA was recovered (Table 1, second versus first row), but it now has RIN values of 7.2 to 7.6, suggesting slight RNA degradation. Electropherograms of cRNA from PAXgene RNA undergoing globin reduction via capture or inhibitory oligo technique resulted in smaller globin peaks compared with no reduction control (Figure 2B). However, the cRNA from the inhibitory oligo method showed smaller size distribution than the other methods. These results indicated that, for high-quality RNA, the globin reduction techniques were mostly efficient but could induce particular changes in RNA quality parameters such as RNA recovery amount, cRNA yield, RIN, and cRNA size distribution.Quality of Microarray MeasurementsThere was no difference in scale factors (SFs) of gene expression signal distributions among the cell line groups, suggesting that the majority of signals were unchanged among groups. Thus, scaled signal values were used throughout unless specified. Graphs of signals of the four spiked-in mRNA controls versus known concentrations showed the expected linear relationship. Background was highest in the Jurkat-globin RNA control group (Table 1). The ratios of signals from the 3′ to the 5′ region of GAPDH and Actin transcripts were all below 3, indicating good RNA quality and efficient in vitro transcription.Similar results were observed for PAXgene RNA samples. Background was lower in the PAXgene RNA control group than in the Jurkat-globin control group, but the 3′/5′ GAPDH and Actin ratios showed a higher trend than the cell line RNA groups (Table 1), probably because of differences in sample type, collection, and handling. No other differences were detected. These results suggest that the microarray measurements were of sufficient quality for inclusion in further statistical analysis.Effects of Globin Reduction on Microarray Detection of TranscriptsJurkat RNA showed the highest percentage of transcript detected, with no differences among globin reduction methods or controls [Table 1, present calls (%)]. For Jurkat-globin RNA, removal of globin mRNA using either the capture or the inhibitory method increased the percentage of transcripts detected by 9 or 5%, respectively, approaching those of Jurkat RNA alone. The capture oligo method resulted in more transcripts detected than the inhibitory oligo method (t-test, P < 0.05).Table 2 shows the cross-tabulation of transcript detection calls between Jurkat-globin RNA with globin removal treatments versus Jurkat RNA alone as the true detection calls. Treatment of Jurkat-globin RNA with the capture or inhibitory oligo method increased concordant present calls by 8 and 5% and decreased false negative calls by 7 and 4% but increased false positive calls only by 1 and 0.22%, respectively, relative to no treatment control. The sensitivities for detection of transcripts from Jurkat-globin RNA after capture, inhibitory, or no treatment were 85.9% [=38.64/(38.64 + 6.33)], 79.3%, and 68.8%, whereas the specificities were 95.3% [=50.0/(2.49 + 50.0)], 96.7%, and 97.1%, respectively. These results suggested that the capture oligo method enabled the highest sensitivity of detection of transcript (t-test, P < 0.01) without significant loss of specificity.Table 2Cross-Tabulation of Detection Calls between Jurkat-Globin RNA with Various Treatments versus Jurkat RNAJurkat-globin RNACapture DNA oligoInhibition PNA oligoControlDetection callsP*Present calls. (%)A†Absent calls. (%)P (%)A (%)P (%)A (%)Jurkat RNAP (%)38.64 ± 0.67‡STDEV.6.33 ± 1.0935.43 ± 0.629.26 ± 1.3930.64 ± 1.2413.88 ± 1.84A (%)2.49 ± 0.4850.00 ± 1.051.72 ± 0.3050.90 ± 1.311.50 ± 0.2351.13 ± 1.36The overall call concordance excluding margin calls between Jurkat and JG RNA was tabulated.* Present calls.† Absent calls.‡ STDEV. Open table in a new tab For PAXgene RNA, removal of globin mRNA increased the percentage of transcripts detected by 6.5% for the capture oligo method and 5.8% for the inhibitory oligo method compared with no treatment control, but with no difference in transcript detection between the two globin reduction methods [Table 1, present calls (%)]. Thus, by comparing the percentages of transcripts detected among various groups and the agreement between transcripts detected from cell line RNA with and without globin, we determined that the globin reduction methods specifically increased transcript detection.Effects of Globin Reduction Techniques on Transcript VariationJurkat RNA with no treatment control showed similar coefficient of variation (CV) as those for Jurkat-globin RNA with no treatment (Figure 3A, JC versus JGC), suggesting that the high amount of spiked globin mRNA did not account for increases in CVs. However, if one unnecessarily performed globin reduction methods on Jurkat RNA alone, then the CV increased (Figure 3A; JC, JP, JD), likely because of the additional steps. The capture oligo method increased the CV more than the inhibitory method probably because of the greater number of steps needed in the former method. On the other hand, if one appropriately performed globin reduction steps with Jurkat-globin RNA, then the CV was lowered compared with those of Jurkat RNA alone undergoing globin reduction (Figure 3A; JGD versus JD, JGP versus JP). These results indicated that the presence of globin mRNA mitigates the increase in CV due to additional processing steps of globin reduction.Figure 3The globin reduction techniques affect on transcript signal variation is dependent on RNA type. The overabundance of globin mRNA mitigates the increase in transcript signal variation because of the additional steps required for globin reduction. Coefficient of variation versus signals from all probe sets for various RNA types with different technical treatments. Curves shown are from Loess 2-degree freedom fitting of the signal data points. A: Cell line RNA. Jurkat (J) or Jurkat-globin (JG) RNA treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). B: PAXgene blood RNA treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Different from the cell line RNA above, PAXgene RNA that underwent the inhibitory method showed slightly higher CV than the other treatments (Figure 3B, BP versus BD and BC). Pearson correlation coefficients among triplicates for each of the nine groups ranged from 0.985 to 0.997 (data not shown) and showed the same trends as the CV graphs in Figure 3. Thus, comparisons of measures of variation suggest that the globin reduction techniques affect transcript signal variation in a manner that is dependent on the type of RNA.Effects of Globin Reduction on Gene Expression ProfilesMultidimensional scaling indicated that triplicates were tightly grouped and Jurkat RNA profiles with different technical conditions clustered close to one another, whereas clusters for Jurkat-globin RNA with different technical conditions were more distanced from each other than those for Jurkat RNA (Figure 4A, J versus JG). In addition, the capture oligo-treated Jurkat-globin RNA cluster located closest to the Jurkat RNA clusters (Figure 4A; JGD and JD, JC, CP). Similar results were observed from hierarchical clustering analysis (Figure 4C). These results suggested that globin removal from Jurkat-globin RNA using the capture oligo method resulted in gene expression profiles most similar to that of Jurkat RNA. PAXgene RNA also formed three clusters of triplicate corresponding to each technical condition, with the triplicates undergoing either globin reduction methods being closer to each other (Figure 4, B and D). These results suggested that the different methods induced distinct gene expression profiles, but globin reduction brought the profiles closer to the original profile than without treatment, particularly with the capture oligo method.Figure 4Different treatments induced distinct gene expression profiles, but globin reduction brought the profiles closer to the original profile than without treatment, particularly with the capture oligo method. Unsupervised cluster analysis of gene expression profiles from various RNA types with different technical treatments. Logged signals from all probe sets were used. A and B: Multidimensional scaling of unscaled signals. C and D: Hierarchical clustering using center correlation and average linkage. In A and C cell line RNA, Jurkat (J) or Jurkat-globin (JG) RNA were treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). In B and D, PAXgene blood RNA was treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Globin Reduction on Specific Transcript SignalsThe HG-U133 plus 2.0 microarray has ∼54,120 probe sets to detect more than 47,000 human transcripts. Class comparison analysis resulted in 8614 probe sets that showed a difference in expression level in at least one of the cell line RNA groups. The signals from these probe sets are shown in the sample clustered heat-map in Figure 5A. Group I 490 probe sets showed differences between Jurkat-globin RNA versus Jurkat RNA alone, regardless of method. Group II represented 3833 probe sets that were similarly at low signal in Jurkat-globin RNA with capture oligo method and all Jurkat RNA but were at higher signals in Jurkat-globin RNA with inhibitory method and at highest signals in Jurkat-globin RNA control. These probe sets probably represent false positives for the inhibitory and control treatments and might be attributable to unspecific binding of globin mRNA to these probes. Group III represented 1476 high signaled probe sets in Jurkat RNA samples but low signals in all Jurkat-globin RNA samples. These false negative probe sets could result from increased background from globin mRNA overabundance. Group IV represented 1380 probe sets that could be revealed after globin RNA reduction with the capture oligo protocol but not the inhibitory protocol or after no treatment. Group V represented 1435 unique higher signal probe sets resulting from the capture oligo protocol and probably represent false positive for this method. Class comparison analysis resulted in 1988 probe sets that showed differences in expression levels among PAXgene RNA treatment groups (Figure 5B). These results more specifically characterized the number of probe sets, the magnitude and direction, and patterns of changes in gene expression profiles induced by RNA types and treatment methods.Figure 5Patterns of changes in differential transcript signals induced by RNA types and treatment methods. Heat-maps of probe sets with differing signals among groups. Probe sets and samples were clustered using center correlation and average linkage. Only dendrograms from clustering of samples are shown on top of each panel. To select from 54,120 probe sets for those with differing signals, analysis of variance with random variance model was performed using P values ≤0.001 as significant. A: Heat map and sample clustering dendrogram of 8614 probe sets with significant differences in signals among the cell line RNA groups. Jurkat (J) or Jurkat-globin (JG) RNA treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). I to V based on JGD expression pattern. B: Heat map and sample clustering dendrogram of 1988 probe sets with differing in signals among the PAXgene blood RNA groups treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionWe have systematically evaluated and compared the effects of two globin reduction methods on gene expression profile measurement outcomes from cell line and PAXgene whole blood RNA. We showed that the globin reduction techniques were mostly efficient at reducing globin mRNA from cRNA but can induce RNA type-dependent changes in quality of RNA during sample preparation and quality of microarray resul
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