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Improved Methods for Gene Expression Profiling from Blood Samples
Ambion offers integrated solutions for gene expression profiling from whole blood, including sample collection and stabilization, blood cell fractionation, RNA isolation, and globin mRNA removal (Figure 1). 70% of the total mRNA in blood is globin mRNA and up to 95% can be removed with these tools. RNA produced by any of these protocols is ready for amplification and subsequent microarray analysis.
Figure 1. Ambion Tools to Aid In Blood Sample RNA Isolation and Expression Profiling.
Expression profiling tools such as microarrays and real-time RT-PCR provide a powerful means of identifying blood-based RNA biomarkers associated with pathological conditions [1–3]. Molecular clues of infection, inflammation, and autoimmune disease are carried in circulating leukocytes, including B-cells, T-cells, neutrophils, monocytes, and other less abundant cell types. However, the large proportion of reticulocyte-derived globin mRNA present in RNA prepared from whole blood can diminish the sensitivity of microarray profiling. The following tools for sample collection and stabilization, blood cell fractionation, RNA isolation, and globin mRNA removal, yield RNA that will perform better in microarray analysis.
Sample Collection and Stabilization
It is beneficial to ensure RNA stabilization by storing blood samples in RNAlater® Tissue Collection:Stabilization Solution. RNAlater Solution protects RNA from degradation and stabilizes the mRNA profile, eliminating the need to immediately isolate the RNA (Figure 2). Treated samples can be safely stored at ambient temperature for up to three days (Figure 3). To facilitate RNA isolation, RNAlater Solution is included in the blood-specific RNA purification products, LeukoLOCK™ Total RNA Isolation System and the RiboPure™-Blood Kit.
Figure 2. RNAlater® Stabilizes a Broader Set of Transcripts Than the PAXgene® System. The ratio of day 0 to day 3 expression for all genes on the Affymetrix® Human Focus Arrays were calculated for blood samples processed with RNAlater Solution and then the RiboPure™-Blood Kit (RNAlater/RiboPure-Blood), and the PAXgene protocol (PAX). (A) The expression ratios were ordered from high to low for PAX samples, along with the corresponding signals from samples processed with RNAlater/RiboPure-Blood. (B) The expression ratios were ordered from high to low for RNAlater/RiboPure-Blood samples, along with the corresponding signals from PAXgene samples.
Figure 3. RNA Stabilized within Blood Samples. Whole blood samples of 0.5 ml, 0.4 ml, and 0.3 ml were collected in EDTA-containing Vacutainer® tubes. Aliquots of each sample were either added directly to 1.3 ml of RNAlater® Solution or lysed directly in 0.8 ml of the Lysis Solution from the RiboPure™-Blood Kit. Samples were stored either at room temperature (RT) or at –20°C for 3 days. RNA was then isolated using the RiboPure-Blood Kit (no DNase treatment) and equal volumes of the RNA recovered were evaluated on a denaturing agarose gel.
Figure 2. RNAlater® Stabilizes a Broader Set of Transcripts Than the PAXgene® System. The ratio of day 0 to day 3 expression for all genes on the Affymetrix® Human Focus Arrays were calculated for blood samples processed with RNAlater Solution and then the RiboPure™-Blood Kit (RNAlater/RiboPure-Blood), and the PAXgene protocol (PAX). (A) The expression ratios were ordered from high to low for PAX samples, along with the corresponding signals from samples processed with RNAlater/RiboPure-Blood. (B) The expression ratios were ordered from high to low for RNAlater/RiboPure-Blood samples, along with the corresponding signals from PAXgene samples.
Figure 3. RNA Stabilized within Blood Samples. Whole blood samples of 0.5 ml, 0.4 ml, and 0.3 ml were collected in EDTA-containing Vacutainer® tubes. Aliquots of each sample were either added directly to 1.3 ml of RNAlater® Solution or lysed directly in 0.8 ml of the Lysis Solution from the RiboPure™-Blood Kit. Samples were stored either at room temperature (RT) or at –20°C for 3 days. RNA was then isolated using the RiboPure-Blood Kit (no DNase treatment) and equal volumes of the RNA recovered were evaluated on a denaturing agarose gel.
RNA Isolation from Whole Blood
RNA quality can have a significant impact on downstream analyses such as array analysis and real-time RT-PCR. To avoid interference from enzymatic inhibitors found in whole blood (e.g., heme) and from anticoagulants used to make analysis of blood samples possible (e.g., heparin), RNA purification from whole blood requires a stringent method that provides high quality RNA depleted of inhibiting agents and contaminants. Ambion provides several blood-specific RNA isolation products, distinguished by the size of the samples to be processed, the need to purify viral RNA or reduce globin mRNA, and the requirement for high throughput isolation.
Small Sample Volumes (0.3–0.5 ml): The RiboPure-Blood Kit purifies RNA directly from whole blood without requiring fractionation of white blood cells. By combining phenol/chloroform extraction, glass fiber filter-based purification, and DNase treatment, the RiboPure-Blood procedure removes protein, heme, genomic DNA, and RNases, resulting in high yields of pure RNA ideal for RT-PCR (Figure 4). The procedure is simple and fast—go from blood to RNA in <30 min. RNAlater Solution is included in the RiboPure-Blood Kit for the stabilization of RNA within the whole blood sample prior to processing for RNA. The kit is also available in custom formats (e.g., for larger sample volumes).
Figure 4. Real Time RT-PCR Analysis of RNA Isolated Using the RiboPure™-Blood Kit. Total RNA isolated from whole blood with the RiboPure-Blood Kit was reverse transcribed and used as template for real-time PCR. Representative results are shown above for amplification of GAPDH, p53, and BTK. Note: Genomic DNA was not detectable in “no RT” control assays.
Large Sample Volumes (3–10+ ml; includes globin depletion): The LeukoLOCK™ Total RNA Isolation System is an innovative method for the fractionation of whole blood, as well as for stabilization and extraction of total RNA from the leukocyte population of whole blood. The system incorporates leukocyte capture filter technology to isolate leukocytes from whole blood, and RNAlater Tissue Collection:Stabilization Solution to stabilize RNA in the cells captured on the filter. After the <5 minute capture and stabilization procedure, leukocytes can be stored directly on the filters for months at -20°C, or shipped at ambient temperature from the point of collection to a second site for RNA extraction. Components for blood fractionation and in-cell RNA stabilization can be obtained separately from the RNA isolation reagents to facilitate multi-site sample processing.
RNA is purified by first disrupting the captured cells in a guanidinium thiocyanate-based solution that releases the RNA while simultaneously inactivating nucleases. This cell lysate is collected and briefly treated with Proteinase K. RNA is then isolated from the lysate using Ambion's MagMAX™ magnetic bead-based technology, followed by optional DNase treatment (with TURBO™ DNase) and final clean-up.
Both quantitative RT-PCR and microarray expression studies have validated the suitability of RNA purified with LeukoLOCK leukocyte capture filters for reliable transcriptome profiling. Samples processed immediately, or fractionated and then stored frozen for 32 days on LeukoLOCK filters, had the same Ct values in qRT-PCR experiments (four different gene targets were measured, including transcripts known to be extremely labile in ex vivo blood; Figure 5). Note that due to removal of red blood cells, RNA purified from the captured leukocytes is depleted of globin mRNA, improving its utility for expression profiling and other applications.
Figure 5. Biotinylated aRNA Amplified from Whole Blood Processed with the LeukoLOCK™ Total RNA Isolation System and a the PAXgene™ Blood RNA System. RNA was isolated from whole blood just after collection (Day 0), or from RNAlater® Solution-treated leukocytes or stabilized blood stored frozen for 32 days (Day 32) prior to isolation using either Ambion’s LeukoLOCK Total RNA Isolation System or the PAXgene Blood RNA System. Samples from each prep (1 ug) were then amplified using the MessageAmp™ II aRNA Amplification Kit to generate biotinylated aRNA. Agilent® 2100 bioanalyzer traces of 1 µl of a 1:10 dilution of of the aRNA generated are shown above.
High Throughput Viral and Total RNA From Blood: The MagMAX™-96 Blood RNA Isolation Kit is designed for rapid (<1 hour), high throughput isolation of total and viral RNA from mammalian whole blood in 96 well plate format (Figure 6). The cells are lysed, and RNA is purified from the lysate using Ambion’s MagMAX™ magnetic bead-based technology. Magnetic beads offer many benefits for isolating RNA from blood. Magnetic beads bind RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields. Furthermore, the RNA yields are more consistent from experiment to experiment, ensuring more reliable results from your downstream analyses. Viral and total RNA obtained using the MagMAX-96 Blood Kit can be used directly in qRT-PCR for viral RNA identification or gene expression profiling.
Figure 6. Consistent Yield, Purity, and Integrity of RNA Isolated from Human Whole Blood with the MagMAX™-96 Blood RNA Isolation Kit. Total RNA was isolated from 50 µl fresh human blood (single donor) in 8 replicates using the MagMAX-96 Blood RNA Isolation Kit. Spectrophotometer readings were obtained by reading 2 µl of the purified RNA on a NanoDrop® ND-1000. The ratio of 28S to 18S rRNA was obtained by analyzing 3 µl of the RNA (~50 ng) on an RNA LabChip® Kit with an Agilent® 2100 bioanalyzer.
Small Sample Volumes (0.3–0.5 ml): The RiboPure-Blood Kit purifies RNA directly from whole blood without requiring fractionation of white blood cells. By combining phenol/chloroform extraction, glass fiber filter-based purification, and DNase treatment, the RiboPure-Blood procedure removes protein, heme, genomic DNA, and RNases, resulting in high yields of pure RNA ideal for RT-PCR (Figure 4). The procedure is simple and fast—go from blood to RNA in <30 min. RNAlater Solution is included in the RiboPure-Blood Kit for the stabilization of RNA within the whole blood sample prior to processing for RNA. The kit is also available in custom formats (e.g., for larger sample volumes).
Figure 4. Real Time RT-PCR Analysis of RNA Isolated Using the RiboPure™-Blood Kit. Total RNA isolated from whole blood with the RiboPure-Blood Kit was reverse transcribed and used as template for real-time PCR. Representative results are shown above for amplification of GAPDH, p53, and BTK. Note: Genomic DNA was not detectable in “no RT” control assays.
Large Sample Volumes (3–10+ ml; includes globin depletion): The LeukoLOCK™ Total RNA Isolation System is an innovative method for the fractionation of whole blood, as well as for stabilization and extraction of total RNA from the leukocyte population of whole blood. The system incorporates leukocyte capture filter technology to isolate leukocytes from whole blood, and RNAlater Tissue Collection:Stabilization Solution to stabilize RNA in the cells captured on the filter. After the <5 minute capture and stabilization procedure, leukocytes can be stored directly on the filters for months at -20°C, or shipped at ambient temperature from the point of collection to a second site for RNA extraction. Components for blood fractionation and in-cell RNA stabilization can be obtained separately from the RNA isolation reagents to facilitate multi-site sample processing.
RNA is purified by first disrupting the captured cells in a guanidinium thiocyanate-based solution that releases the RNA while simultaneously inactivating nucleases. This cell lysate is collected and briefly treated with Proteinase K. RNA is then isolated from the lysate using Ambion's MagMAX™ magnetic bead-based technology, followed by optional DNase treatment (with TURBO™ DNase) and final clean-up.
Both quantitative RT-PCR and microarray expression studies have validated the suitability of RNA purified with LeukoLOCK leukocyte capture filters for reliable transcriptome profiling. Samples processed immediately, or fractionated and then stored frozen for 32 days on LeukoLOCK filters, had the same Ct values in qRT-PCR experiments (four different gene targets were measured, including transcripts known to be extremely labile in ex vivo blood; Figure 5). Note that due to removal of red blood cells, RNA purified from the captured leukocytes is depleted of globin mRNA, improving its utility for expression profiling and other applications.
Figure 5. Biotinylated aRNA Amplified from Whole Blood Processed with the LeukoLOCK™ Total RNA Isolation System and a the PAXgene™ Blood RNA System. RNA was isolated from whole blood just after collection (Day 0), or from RNAlater® Solution-treated leukocytes or stabilized blood stored frozen for 32 days (Day 32) prior to isolation using either Ambion’s LeukoLOCK Total RNA Isolation System or the PAXgene Blood RNA System. Samples from each prep (1 ug) were then amplified using the MessageAmp™ II aRNA Amplification Kit to generate biotinylated aRNA. Agilent® 2100 bioanalyzer traces of 1 µl of a 1:10 dilution of of the aRNA generated are shown above.
High Throughput Viral and Total RNA From Blood: The MagMAX™-96 Blood RNA Isolation Kit is designed for rapid (<1 hour), high throughput isolation of total and viral RNA from mammalian whole blood in 96 well plate format (Figure 6). The cells are lysed, and RNA is purified from the lysate using Ambion’s MagMAX™ magnetic bead-based technology. Magnetic beads offer many benefits for isolating RNA from blood. Magnetic beads bind RNA more efficiently than glass fiber filter methods, resulting in higher RNA yields. Furthermore, the RNA yields are more consistent from experiment to experiment, ensuring more reliable results from your downstream analyses. Viral and total RNA obtained using the MagMAX-96 Blood Kit can be used directly in qRT-PCR for viral RNA identification or gene expression profiling.
Figure 6. Consistent Yield, Purity, and Integrity of RNA Isolated from Human Whole Blood with the MagMAX™-96 Blood RNA Isolation Kit. Total RNA was isolated from 50 µl fresh human blood (single donor) in 8 replicates using the MagMAX-96 Blood RNA Isolation Kit. Spectrophotometer readings were obtained by reading 2 µl of the purified RNA on a NanoDrop® ND-1000. The ratio of 28S to 18S rRNA was obtained by analyzing 3 µl of the RNA (~50 ng) on an RNA LabChip® Kit with an Agilent® 2100 bioanalyzer.
Removing Globin mRNA from Blood Samples
Array analyses using whole blood total RNA containing a high percentage of globin mRNA has been shown to decrease Present Calls, decrease call concordance, and increase signal variation [4]. Thus, globin mRNA interferes with the detection of other, less abundant genes, and data can be improved if the globin transcripts are removed.
The GLOBINclear™-Human and GLOBINclear-Mouse/Rat Kits employ subtractive hybridization technology to remove >95% of the globin mRNA from whole blood total RNA. The magnetic bead based procedure leverages the strength of biotin/streptavidin binding, the specificity of nucleic acid hybridization, and the convenience of magnetic bead separation to deplete globin mRNA from blood total RNA. Because there is no RNase H treatment, the resulting RNA is a superior template for RNA amplification or for synthesizing cDNA for array analysis and quantitative RT-PCR. When used for RNA labeling/amplification for array analysis, RNA from Ambion's GLOBINclear Systems delivers a significant increase in sensitivity and a concomitant drop in variability, relative to untreated whole blood RNA. As an example, the array analysis results in Figure 7 show up to 50% more gene features called Present—the equivalent of thousands of genes—in the GLOBINclear-treated blood samples versus untreated blood samples. Figure 7 also demonstrates that GLOBINclear-processed RNA is ideal for use with Ambion’s MessageAmp™ II aRNA Amplification Kits.
Figure 7. GLOBINclear™-Human Treatment of Whole Blood RNA Increases the Number of Genes Detected in Array Analysis. Duplicate arrays (Affymetrix® GeneChip® U133 Plus 2.0) were hybridized with two GLOBINclear-treated whole blood total RNA samples for each donor shown. Duplicate arrays were also hybridized with untreated total RNA samples from each donor. GLOBINclear-treated samples demonstrated a clear and consistent increase in features called Present (average increase of 50%). Ambion’s MessageAmp™ II-96 aRNA Amplification Kit was used to prepare biotinylated aRNA for hybridization using 1 µg input RNA.
The GLOBINclear™-Human and GLOBINclear-Mouse/Rat Kits employ subtractive hybridization technology to remove >95% of the globin mRNA from whole blood total RNA. The magnetic bead based procedure leverages the strength of biotin/streptavidin binding, the specificity of nucleic acid hybridization, and the convenience of magnetic bead separation to deplete globin mRNA from blood total RNA. Because there is no RNase H treatment, the resulting RNA is a superior template for RNA amplification or for synthesizing cDNA for array analysis and quantitative RT-PCR. When used for RNA labeling/amplification for array analysis, RNA from Ambion's GLOBINclear Systems delivers a significant increase in sensitivity and a concomitant drop in variability, relative to untreated whole blood RNA. As an example, the array analysis results in Figure 7 show up to 50% more gene features called Present—the equivalent of thousands of genes—in the GLOBINclear-treated blood samples versus untreated blood samples. Figure 7 also demonstrates that GLOBINclear-processed RNA is ideal for use with Ambion’s MessageAmp™ II aRNA Amplification Kits.
Figure 7. GLOBINclear™-Human Treatment of Whole Blood RNA Increases the Number of Genes Detected in Array Analysis. Duplicate arrays (Affymetrix® GeneChip® U133 Plus 2.0) were hybridized with two GLOBINclear-treated whole blood total RNA samples for each donor shown. Duplicate arrays were also hybridized with untreated total RNA samples from each donor. GLOBINclear-treated samples demonstrated a clear and consistent increase in features called Present (average increase of 50%). Ambion’s MessageAmp™ II-96 aRNA Amplification Kit was used to prepare biotinylated aRNA for hybridization using 1 µg input RNA.
Summary
Blood is the most commonly used tissue for clinical research. Unfortunately, blood samples present obstacles to gene expression analyses that are not encountered with most other tissues. The complex cellular nature of blood makes it difficult to detect differential gene expression that occurs in only a subset of cell types. Furthermore, globin mRNA makes up the majority of mRNA in whole blood total RNA samples, thus diminishing the sensitivity of expression profiling for other, less abundant RNA species. Ambion provides dedicated tools for blood analysis. Not only do these tools address stabilization of RNA within blood samples, making it easier to collect samples in the field, but they are optimized to remove the unique contaminants found in blood, including red blood cells (using the LeukoLOCK™ system) or abundant globin transcripts (using GLOBINclear™ Kits ), rendering white blood cell expression profiling more meaningful.
Scientific Contributors
Mangkey Bounpheng, Angela Burrell, Kurt Evans, Xingwang Fang, Marianna Goldrick, Juanita C. Gonzales, Nathan Harris, Jennifer Ho, Quoc Hoang, Jon Kemppanien, Gary Latham, Ivonne Moon, Sharmili Moturi, Bob Setterquist, Penn Whitley, Chris Willis, WeiWei Xu • Ambion, Inc.
Scientific Contributors
Mangkey Bounpheng, Angela Burrell, Kurt Evans, Xingwang Fang, Marianna Goldrick, Juanita C. Gonzales, Nathan Harris, Jennifer Ho, Quoc Hoang, Jon Kemppanien, Gary Latham, Ivonne Moon, Sharmili Moturi, Bob Setterquist, Penn Whitley, Chris Willis, WeiWei Xu • Ambion, Inc.
References1. Burczynski ME et al. (2005) Transcriptional profiles in peripheral blood mononuclear cells prognostic of clinical outcomes in patients with advanced renal cell carcinoma. Clin Cancer Res 11(3):1181–9.
2. Cobb JP et al. (2005) Application of genome-wide expression analysis to human health and disease. Proc Natl Acad Sci USA 102(13):4801–6.
3. Tsuang MT, Nossova N, Yager T, Tsuang MM, Guo SC, Shyu KG, Glatt SJ, Liew CC (2005) Assessing the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder: a preliminary report. Am J Med Genet B Neuropsychiatr Genet 133(1):1–5.
4. Feezor RJ et al. (2004) Whole blood and leukocyte RNA isolation for gene expression analyses. Physiol Genomics 19:247–54.
2. Cobb JP et al. (2005) Application of genome-wide expression analysis to human health and disease. Proc Natl Acad Sci USA 102(13):4801–6.
3. Tsuang MT, Nossova N, Yager T, Tsuang MM, Guo SC, Shyu KG, Glatt SJ, Liew CC (2005) Assessing the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder: a preliminary report. Am J Med Genet B Neuropsychiatr Genet 133(1):1–5.
4. Feezor RJ et al. (2004) Whole blood and leukocyte RNA isolation for gene expression analyses. Physiol Genomics 19:247–54.