A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing Buffer
2015; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2014.11.002
ISSN1943-7811
AutoresChang Hee Kim, Majid Abedi, Yenbou Liu, Sree Panuganti, Francisco Flores, Kevin R. Shah, Hannah Catterall, Krishna S. Morampudi, Robert Terbrueggen,
Tópico(s)Gene expression and cancer classification
ResumoWe describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification. We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification. Genome level screening technologies such as microarrays and whole transcriptome sequencing are being used to discover gene expression signatures that may improve the diagnosis of disease, predict therapeutic response, and determine disease prognosis.1Chibon F. Cancer gene expression signatures - the rise and fall?.Eur J Cancer. 2013; 49: 2000-2009Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Although a number of gene expression signature tests are now in clinical development2Nielsen T. Wallden B. Schaper C. Ferree S. Liu S. Gao D. Barry G. Dowidar N. Maysuria M. Storhoff J. Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens.BMC Cancer. 2014; 14: 177Crossref PubMed Scopus (218) Google Scholar, 3Braza F. Soulillou J.P. Brouard S. Gene expression signature in transplantation tolerance.Clin Chim Acta. 2012; 413: 1414-1418Crossref PubMed Scopus (15) Google Scholar and a few examples have been cleared by the US Food and Drug Administration for in vitro diagnostic use,4Sabatier R. Goncalves A. Bertucci F. Personalized medicine: present and future of breast cancer management.Crit Rev Oncol Hematol. 2014; 91: 223-233Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar the routine clinical use of gene signature tests is limited by the lack of large-scale clinical validation studies and the complexity and high cost of performing these multiplexed tests. We have developed a method that combines an RNA-stabilizing buffer and the target-dependent chemical ligation of probes, followed by PCR amplification of the ligated probes to perform the quantitative analysis of multiple transcripts directly from blood samples without the requirement for RNA purification. Unlike enzymatic ligation, the chemical ligation reaction is not inhibited by sample contaminants. The method, termed chemical ligation-dependent probe amplification (CLPA), is performed in a single reaction tube, and the final assay products can be analyzed with existing capillary electrophoresis (CE)-based DNA-sequencing instruments. We have selected a set of 10 genes, 7 of which are responsive to ionizing radiation (response genes), and 3 of which are normalization genes (Table 1), to demonstrate the performance of CLPA. Changes in the expression of the response genes relative to the normalizer genes were measured in peripheral blood before and after ex vivo exposure to ionizing radiation; such changes have been well documented in the literature.5Paul S. Amundson S.A. Development of gene expression signatures for practical radiation biodosimetry.Int J Radiat Oncol Biol Phys. 2008; 71: 1236-1244Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Kabacik S. Mackay A. Tamber N. Manning G. Finnon P. Paillier F. Ashworth A. Bouffler S. Badie C. Gene expression following ionising radiation: identification of biomarkers for dose estimation and prediction of individual response.Int J Radiat Biol. 2011; 87: 115-129Crossref PubMed Scopus (113) Google Scholar, 7Meadows S.K. Dressman H.K. Muramoto G.G. Himburg H. Salter A. Wei Z. Ginsburg G.S. Chao N.J. Nevins J.R. Chute J.P. Gene expression signatures of radiation response are specific, durable and accurate in mice and humans.PLoS One. 2008; 3: e1912Crossref PubMed Scopus (92) Google Scholar, 8Dressman H.K. Muramoto G.G. Chao N.J. Meadows S. Marshall D. Ginsburg G.S. Nevins J.R. Chute J.P. Gene expression signatures that predict radiation exposure in mice and humans.PLos Med. 2007; 4: e106Crossref PubMed Scopus (146) Google Scholar, 9Amundson S.A. Do K.T. Shahab S. Bittner M. Meltzer P. Trent J. Fornace Jr., A.J. Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation.Rad Res. 2000; 154: 342-346Crossref PubMed Scopus (242) Google ScholarTable 1Radiation Responsive and Normalization Genes Used in the Multiplex Probe SetGene nameGene symbolRefSeq ID∗Accession numbers from http://www.ncbi.nlm.nih.gov/nuccore.Ligated product length (bp)Mitochondrial ribosomal protein S5MRPS5NM_031902115v-myc avian myelocytomatosis viral oncogene homologMYCNM_002467120Cyclin-dependent kinase inhibitor 1ACDKN1ANM_000389125Cerebellar degeneration-related protein 2CDR2NM_001802135BCL2-associated X proteinBAXNM_138761143Ferredoxin reductaseFDXRNM_024417148BCL2 binding component 3BBC3NM_001127240155Glyceraldehyde-3-phosphate dehydrogenaseGAPDHNM_002046161Mitochondrial ribosomal protein S18AMRPS18ANM_018135165Proliferating cell nuclear antigenPCNANM_002592180∗ Accession numbers from http://www.ncbi.nlm.nih.gov/nuccore. Open table in a new tab The CLPA methodology relies on the nonenzymatic, chemical ligation of adjacently hybridized DNA oligonucleotides to form specific, uniquely sized ligation products in direct proportions to the nucleic acid targets. The method uses chemically reactive groups at either the 5′ or 3′ end of the ligation probes. The sulfur (S)-probe contains a 3′-phosphorothioate group, and the leaving (L)-probe contains a 5′-dimethylaminoazobenzenesulfonyl leaving group on a modified T base10Abe H. Kool E.T. Universal linkers for signal amplification in auto-ligating probes.Nucleic Acids Symp Ser (Oxf). 2005; 126: 37-38Crossref Scopus (3) Google Scholar, 11Xu Y. Karalkar N.B. Kool E.T. Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations.Nat Biotechnol. 2001; 19: 148-152Crossref PubMed Scopus (142) Google Scholar, 12Sando S. Kool E.T. Nonenzymatic DNA ligation in Escherichia coli cells.Nucleic Acids Res Suppl. 2002; 2: 121-122Crossref PubMed Scopus (3) Google Scholar, 13Sando S. Kool E.T. Quencher as leaving group: efficient detection of DNA-joining reactions.J Am Chem Soc. 2002; 124: 2096-2097Crossref PubMed Scopus (108) Google Scholar, 14Sando S. Kool E.T. Imaging of RNA in bacteria with self-ligating quenched probes.J Am Chem Soc. 2002; 124: 9686-9687Crossref PubMed Scopus (119) Google Scholar (Figure 1). Each probe contains three primary domains: a target sequence that is complementary to the RNA target and determines specificity, a universal forward or reverse primer sequence for robust multiplex amplification, and spacer sequences (one or two) that are varied in length such that each target-specific ligation product is uniquely sized and separated in length by at least four base pairs. All S- and L- probes contain either a universal forward or reverse primer to allow for multiplex PCR amplification of all ligation products with either one or two universal PCR primer sets, depending on the number of targets to be detected. The individual probes themselves cannot be amplified, because they contain only one of the two required primer sequences. The chemical ligation reaction occurs when a probe pair hybridizes to adjacent sequences on the RNA target, resulting in displacement of the 5′-dimethylaminoazobenzenesulfonyl leaving group by the nucleophilic phosphorothioate group and formation of bond between the two probes. In addition to the S- and L-probes, biotin-labeled target capture (TC)-probes are used to bind the target RNA adjacent to the sequence recognized by the target sequence. The TC-probes facilitate the capture of the complex that contains a ligated probe pair bound to its RNA target with the use of streptavidin-labeled paramagnetic beads. The CLPA assay was performed in five basic steps (Figure 1). Unless otherwise stated, all reagents were provided by DxTerity Diagnostics (Rancho Dominguez, CA) (Supplemental Table S1). RNA in whole blood was stabilized by adding RNA stabilization buffer (DxCollect), which lyzes the cells and stabilizes the RNA. Fifty microliters of stabilized blood or cultured blood (1:2 ratio of blood to stabilization buffer) was mixed in a 96-well plate (Corning Inc., Union City, CA) or a 32-well plate (Axygen Scientific/Corning Inc., Union City, CA) with 15 μL of reaction buffer: 15 μL of a solution that contained S-probes (probe mix A) (Table 2), 15 μL of solution that contained L- and TC-probes (probe mix B) (Table 3), and 5 μL of a protein digestion solution (mix C). Probe mix A contained S-probes and attenuation S (SA)-probes at the concentrations listed in Table 2. Diluent was 1 mmol/L DTT in 1× TE buffer. Probes were heat activated for 2 minutes at 95°C after formulation. Probe mix B contained L- and TC-probes at the concentrations listed in Table 3. The diluent was 1× TE buffer.Table 2Formulation of S-Probes and Sequences in Probe Mix AGeneConcentration∗Concentrations for S-probes were measured in amol/L; concentration for SA-probes was measured in pmol/L.Primer sequenceS-probe (contains 3′ phosphorothioate modification) BAX333.45′-GGGTTCCCTAAGGGTTGGACGCGTTCTAAACGGACTGTTACCAGAGTCTGTGTCCACGGCGGCAATCATCCTC-3′ BBC31333.45′-GGGTTCCCTAAGGGTTGGACGCGTTCTAAATGTACAGAAAATTCATTCCGGTATCTACAGCAGCGCATA-3′ CDKN1A2666.85′-GGGTTCCCTAAGGGTTGGACGCGTCATGCCCTGTCCATAGCCTCTACTGCCACCATC-3′ CDR2666.75′-GGGTTCCCTAAGGGTTGGACGCGGCAACTAAAGATCTCCTTAAACAACGCTTTGTATTCTGGAGG-3′ FDXR2666.85′-GGGTTCCCTAAGGGTTGGACGCGACTCAGTGGAAACAGGCCATTAGACAGATGACCCTCCACAGTCCAGCAGTAGAGAGATGGG-3′ GAPDH205′-GGGTTCCCTAAGGGTTGGACGCGTGGCGAGAGTGTCTCGTATCTCGCTCCTGGAAGATGGTGATGGGATT-3′ MRPS18A2666.85′-GGGTTCCCTAAGGGTTGGACGCGTTCTAAACGGACTGTTACCAGGATGAACTGGCTAAGCAGCAGAACATCGTCA-3′ MRPS51333.45′-GGGTTCCCTAAGGGTTGGACGGTGCAGTCTTCACATCTTCCCAGTCCAGTTTGACG-3′ MYC333.45′-GGGTTCCCTAAGGGTTGGACGCGTGTTCGGTTGTTGCTGATCTGTCTCAGGACTCTGACAC-3′ PCNA666.75′-GGGTTCCCTAAGGGTTGGACGCGTTCTAAACGGACTGTTACCACTTCACCGCAATTTTATACTCTACAACAAGGGGTACATCTGCAGACA-3′SA-probe (contains 3′ phosphorothioate modification) GAPDH1313.45′-CGTGGCGAGAGTGTCTCGTATCTCGCTCCTGGAAGATGGTGATGGGATT-3′S, sulfur; SA, attenuation S.∗ Concentrations for S-probes were measured in amol/L; concentration for SA-probes was measured in pmol/L. Open table in a new tab Table 3Formulation of L- and TC-Probes and Their Respective Sequences in Probe Mix BGeneConcentration (pmol/L)Primer sequenceL-probe (contains 5′-dimethylaminoazobenzenesulfonyl modification) BAX333.45′-TGCAGCTCCATGTTACTGTCCAGTTCGTCCCCACAGGATGAGCCTGCTCTAGATTGGATCTTGCTGGCAC-3′ BBC31333.45′-TACAGTATCTTACAGGCTGGGCCATCCCTCCCCACAGGATGAGCCTTGGAATGTCGGAAATGCTCTAGATTGGATCTTGCTGGCAC-3′ CDKN1A2666.85′-TTAAAATGTCTGACTCCTTGTTCCGCTGCTAATCTGGCGAGAGGCTCTAGATTGGATCTTGCTGGCAC-3′ CDR2666.75′-TGTTGTAGGGGAACTCACGGGCTCTGGGTTGTTCTAAACGGACTGGCTCTAGATTGGATCTTGCTGGCAC-3′ FDXR2666.85′-TAAGGGGTTAGATCGGCCCACACCTCCACCTTGGCGAGAGCTCTAGATTGGATCTTGCTGGCAC-3′ GAPDH1333.45′-TCCATTGATGACAAGCTTCCCGTTCTCAGCTGGACTCAGTGGAAACAGGCCATTAGACAGAACAGGGCTCTAGATTGGATCTTGCTGGCAC-3′ MRPS18A2666.85′-TAGTTATACTTGTGCTTCAGGTTCCAACGGCAGATGGACAGGATGAGCCTTGGAATGTCGGAAATGCTCTAGATTGGATCTTGCTGGCAC-3′ MRPS51333.45′-TCTGGAACCTCATCTTCTGGCTCTGGATCCTTCCGCTCTAGATTGGATCTTGCTGGCAC-3′ MYC333.45′-TGTCCAACTTGACCCTCTTGGCAGCAGGATAGTCGCTCTAGATTGGATCTTGCTGGCAC-3′ PCNA666.75′-TACTGAGTGTCACCGTTGAAGAGAGTGGAGTGGCACAGGATGAGCCTTGGAATGTCGGAAATAGGGCTCTAGATTGGATCTTGCTGGCAC-3′TC-probe (biotinylated) MRPS52666.85′-GGGACGCAACCACAATGGGCAGAGGGC-3′ MRPS52666.85′-GCGGCTCTCTTCAAATTAGACCACACAGAGCGC-3′ MYC2666.85′-GAGTGGAGGGAGGCGCTGCGTAGTTGTGCT-3′ MYC2666.85′-ATTCTCCTCGGTGTCCGAGGACCTGGGGCTG-3′ CDKN1A2666.85′-GCAATGAACTGAGGAGGGATGAGGTGGATGAGGA-3′ CDKN1A2666.85′-GGAAAGACAACTACTCCCAGCCCCATATGAGCCCA-3′ CDR22666.85′-GGCCAGTTCCCAGCCGCTGGCAACAGGCTCAGAC-3′ CDR22666.85′-TGTTCTCTGTTCATCTATTTCCTGCTTAGTTTTC-3′ BAX2666.85′-GCTTGAGACACTCGCTCAGCTTCTTGGTGGAC-3′ BAX2666.85′-GAAAACATGTCAGCTGCCACTCGGAAAAAGACCTCTC-3′ FDXR2666.85′-GGTTACCTCAGTTGCTGAAAGCTAAAACCTTGCGCGAAAAA-3′ FDXR2666.85′-TTTCTTGGTTGCAGCTGTTTTATTTCCAGCATGTTCCCAA-3′ BBC32666.85′-CAGACTCCTCCCTCTTCCGAGATTTCCCACCCTC-3′ BBC32666.85′-GGAAACATACAAAAATCATGTACAAAAAAAATTAACC-3′ GAPDH2666.85′-CGGTGCCATGGAATTTGCCATGGGTGGAATCATA-3′ GAPDH2666.85′-GTACTCAGCGCCAGCATCGCCCCACTTGATTTTGG-3′ MRPS18A2666.85′-GGCCAGAGGGGTTAGGAGGATTTGGACTCTCC-3′ MRPS18A2666.85′-CTGTGATCTTTCGGGGCAGCATGCCTCCATG-3′ PCNA2666.85′-TAAAGAAGTTCAGGTACCTCAGTGCAAAAGTTAG-3′ PCNA2666.85′-ATCCTCGATCTTGGGAGCCAAGTAGTATTTTAAGTGTCCC-3′L, leaving; TC, target capture. Open table in a new tab S, sulfur; SA, attenuation S. L, leaving; TC, target capture. Either four or five technical replicates were performed (as indicated) in all experiments. The plates were sealed with 8-well strip caps (Agilent Technologies, Santa Clara, CA) and incubated in a thermocycler (Veriti; Life Technologies, Carlsbad, CA) for 5 minutes at 55°C, followed by 10 minutes at 80°C and then 2 hours and 45 minutes at 55°C for the chemical ligation step. Because the probes are in excess over the target RNA concentration, the amount of each ligation product formed in the reaction is proportional to the amount of target RNA. After completion of the chemical ligation reaction, 5 μL of 2.7-μm diameter streptavidin-coated paramagnetic beads were added to each well and mixed by pipetting. The samples were then incubated for an additional 15 minutes at 55°C to allow binding of the ligation complex to the beads. The plate was removed from the thermal cycler and placed on a 96-well Side Skirted Magnetic Particle Concentrator (Invitrogen, Carlsbad, CA) for 2 minutes to capture the beads to the side of the well. The liquid reaction mixture was aspirated with a multichannel pipette (Rainin, Columbus, OH). The beads were washed three times with 180 μL of wash solution for bead washing steps, and the wash buffer was removed. The ligation products were amplified by PCR without removal from the paramagnetic beads. One PCR primer of each pair was labeled with a fluorescent dye. A solution that contained Taq DNA polymerase, PCR buffer, and dNTPs (Taq solution) and the universal primer mix [600 nmol/L of forward 5′-(5′ fluorescein)GGGTTCCCTAAGGGTTG-3′ and reverse 5′-GTGCCAGCAAGATCCAATCT-3′ PCR primers] were added to the washed beads, and the mixture was amplified by PCR (2 minutes at 95°C, followed by 30 cycles of 10 seconds at 95°C, 20 seconds at 57°C, and 20 seconds at 72°C). The amounts of each target-specific amplicon remained proportional to the amount of the specific target RNA, because the amplification used a single pair of universal PCR primers. The PCR products were separated by CE and were detected by fluorescence. The PCR product from each target sequence was identified on the basis of its characteristic length; thus, each was quantified independently. A 2-μL aliquot of the final amplified CLPA reaction was mixed with 17.5 μL of Hi-Di Formamide (Life Technologies) and 0.5 μL of GeneScan 600 Liz V2 dye Size Standard (Life Technologies) and injected into a 24-capillary array with POP-6 polymer running on a ABI 3500xL Dx Genetic Analyzer with the Fragment Analysis Module (Life Technologies) according to the manufacturer's guidelines. The standard injection time was 15 seconds (at 18 kV) but was decreased to avoid saturating signals for a few samples according to the manufacturer's recommendations. Blood samples were collected by venipuncture at Access Biologicals (Vista, CA) under a protocol approved by the Schulman Associates institutional review board (Cincinnati, OH, and Fort Lauderdale, FL). After signed informed consents were obtained, samples were collected from six healthy volunteer donors on the same day by two different phlebotomists. A total of 12 tubes of blood were collected from each donor: nine into DxCollect Blood Collection Tubes each with a 1-mL draw into 2 mL of DxCollect buffer (DxTerity Diagnostics) and three into PAXgene RNA Collection Tubes (2.5-mL draw; PreAnalytix GmbH, Hombrechtikon, Switzerland). On collection, all tubes were mixed by inverting 10 times. All blood tubes were immediately shipped at ambient temperature and were received at DxTerity within 24 hours of blood draw. On receipt, three DxCollect tubes and one PAXgene tube per donor were frozen at −20°C. The remaining tubes were split into two groups and incubated for either 7 or 14 days at ambient temperature (15°C to 30°C) at which point the tubes were frozen at −20°C until tested. For total RNA extraction from DxCollect-stabilized blood samples, a 1.0-mL aliquot of blood lysate was removed from each tube for processing. Before performing the extraction procedure, the total RNA was precipitated by first transferring 150 μL of stabilized blood into a nuclease-free 1.7-mL microfuge tube to which 50 μL of DxCollect RNA Precipitation Solution (DxTerity) was added. The resulting solution was mixed by vortexing for 15 seconds and precipitated by centrifugation at 13,800 × g in a microcentrifuge for 15 minutes at 4°C. The supernatant fluid was then discarded, the RNA pellet was resuspended in Norgen Lysis Buffer, and the purification was completed with the Norgen Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada). RNA was isolated from the blood samples collected into PAXgene tubes according to the manufacturer's directions (PreAnalytiX GmbH). All extracted RNA samples were analyzed for RNA concentration and RNA Integrity Number (RIN) scores by using the Agilent 2100 Bioanalyzer RNA Nano kit (Agilent Technologies) according to the manufacturer's directions (Agilent 2100 Bioanalyzer 2100 Expert User's Guide). RNA purity was also determined by taking triplicate measurements of A260/A280 ratios with the use of a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE). A ratio of approximately 2 was considered pure RNA, whereas a ratio appreciably lower than 2 was an indication of the presence of proteins, phenol, or other contaminants. Human blood samples for ex vivo irradiation and culture were obtained from healthy volunteer subjects. De-identified healthy human donor blood samples (six donor samples, approximately 10 mL each) from Hemacare Corporation (Van Nuys, CA) were collected in green-topped sodium heparin-containing Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) and transported at ambient temperature within 3.5 hours to the University of California, Los Angeles, School of Medicine, Department of Pathology and Laboratory Medicine. A 3.0-mL sample of the whole blood was transferred to a T25 flask (Gibco/Life Technologies, Carlsbad, CA) which was then irradiated to 2 Gy by using a cesium-137 gamma-ray source at 4 Gy/min (Mark IV cesium-137 sealed source irradiator). A second sample of 3.0 mL from the primary tube was handled in the same manner but without irradiation. After irradiation or mock exposure, 3.0 mL of RPMI 1640 medium (Gibco/Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT) and 1% penicillin/streptomycin (HyClone) was added to the individual blood samples in the T25 flasks. The blood sample was incubated for 24 hours (±1 hour) at 37°C in a humidified incubator with 5% CO2. After the incubation, 12.0 mL of DxCollect buffer (room temperature) was added to each T25 flask, and the solutions were mixed by swirling the flasks 8 to 10 times. The samples in the T25 flasks were stored in a refrigerator at 2°C to 8°C until transported at ambient temperature to DxTerity Diagnostics where they were stored at 2°C to 8°C before testing by CLPA or TaqMan (Life Technologies, Carlsbad, CA) assay. A 10-plex assay comprising seven radiation responsive genes5Paul S. Amundson S.A. Development of gene expression signatures for practical radiation biodosimetry.Int J Radiat Oncol Biol Phys. 2008; 71: 1236-1244Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Kabacik S. Mackay A. Tamber N. Manning G. Finnon P. Paillier F. Ashworth A. Bouffler S. Badie C. Gene expression following ionising radiation: identification of biomarkers for dose estimation and prediction of individual response.Int J Radiat Biol. 2011; 87: 115-129Crossref PubMed Scopus (113) Google Scholar, 7Meadows S.K. Dressman H.K. Muramoto G.G. Himburg H. Salter A. Wei Z. Ginsburg G.S. Chao N.J. Nevins J.R. Chute J.P. Gene expression signatures of radiation response are specific, durable and accurate in mice and humans.PLoS One. 2008; 3: e1912Crossref PubMed Scopus (92) Google Scholar, 8Dressman H.K. Muramoto G.G. Chao N.J. Meadows S. Marshall D. Ginsburg G.S. Nevins J.R. Chute J.P. Gene expression signatures that predict radiation exposure in mice and humans.PLos Med. 2007; 4: e106Crossref PubMed Scopus (146) Google Scholar, 9Amundson S.A. Do K.T. Shahab S. Bittner M. Meltzer P. Trent J. Fornace Jr., A.J. Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation.Rad Res. 2000; 154: 342-346Crossref PubMed Scopus (242) Google Scholar and three normalizer genes was developed (Table 1). CLPA probe sets were designed with the NEAT Module of the AlleleID software version 7.81 (Premier Biosoft International, Palo Alto, CA). Each gene was designed to have a unique ligated length that could be differentially detected on a CE instrument. Probe sequences and concentrations are listed in Tables 2 and 3, respectively. The sequences of the competitor oligonucleotide SA-probe for GAPDH which are used in all of the experiments is given in Table 2. All universal forward and reverse PCR primers were manufactured at Eurofins Genomics (Huntsville, AL). The universal forward primer was modified to include a 5′ fluorescein dye label. Both primers were used to formulate the primer mix for the amplification step in CLPA. The CE electropherogram data files were processed with GeneMarker software version 2.4.0 (SoftGenetics, State College, PA) to generate the peak height relative fluorescence unit (RFU) values. The peak data tables were saved as .txt files and were analyzed with JMP software version 11.0 (SAS Institute, Cary, NC). Gene normalized values were generated by dividing the RFU values obtained from the instrument by the geometric mean of the RFU values of the MRPS5 and MRPS18A genes from the same sample. In the case of the ex vivo-irradiated blood samples, normalization was performed with the GAPDH housekeeping gene because it was the normalization gene used for the TaqMan quantitative real-time RT-PCR (RT-qPCR) data. DxCollect blood was collected from three healthy human donors. Two-fold serial dilutions of each stabilized blood sample were performed with leukocyte-depleted human blood (Golden West Biologicals, Temecula, CA) as the dilution matrix. Diluted blood (50 μL per donor) was used to perform CLPA assays with each of the five technical replicates per dilution. The final volume of stabilized blood used in the CLPA assay ranged from 50 to 0.78 μL, which corresponds to 16.7 to 0.25 μL of whole blood. Plasmids that contained full-length human cDNA clones for four genes (MRPS18A, MRPS5, PCNA, and FDXR) were obtained from Origene Inc. (Rockville, MD). The RefSeq ID and the catalog numbers are listed in Table 4. The cDNA insert for each gene was amplified from the plasmid by using common primers that flanked the insert site (forward primer, 5′-AATGGGCGGTAGGCGTGTA-3′; reverse primer, 5′-GGAGGGGTCACAGGGATGC-3′; IDT, San Jose, CA). The resulting PCR amplicons were quantified with an Agilent 2100 Bioanalyzer (Agilent Technologies) by using the DNA 1000 assay. RNA transcripts were generated from the PCR-amplified cDNA fragment by using the AmpliScribe T7-Flash Kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer's instructions. Briefly, 100 ng of amplicon (in 40 μL of final volume) was incubated for 45 minutes at 37°C in the AmpliScribe Transcription Reaction Mix, followed by adding RNase-free DNase to remove the DNA template. The resulting RNA was quantified with the Agilent 2100 Bioanalyzer.Table 4Origene Plasmid Clones Used to Generate in Vitro-Transcribed RNA for Figure 5Gene nameRefSeq ID∗Accession numbers from http://www.ncbi.nlm.nih.gov/nuccore.Origene catalog numberMRPS5NM_031902SC107180MRPS18ANM_018135SC113696PCNANM_002592SC118528FDXRNM_024417SC309756∗ Accession numbers from http://www.ncbi.nlm.nih.gov/nuccore. Open table in a new tab In vitro transcripts for each of the four genes were diluted with 1× TE buffer and combined into 12 separate mixes. In each of the 12 in vitro transcript solutions, the concentrations of MRPS18A and MRPS5 mRNA were held constant to result in final in-assay concentrations of 6 and 12 fmol/L, respectively. The concentrations of PCNA and FDXR transcripts were changed by performing 11 threefold serial dilutions (into 1× TE buffer), beginning at a concentration equivalent to a final in-assay concentration of 300 fmol/L. The final concentrations of PCNA and FDXR ranged from 300 fmol/L to 1.7 amol/L. The final (12th) mix did not contain any PCNA or FDXR mRNA. RNA solution (100 μL each) was combined with 200 μL of DxCollect buffer. RNA-DxCollect solution (50 μL each) was used as sample in a CLPA reaction. Assays were performed with five technical replicates. A mix of S-probes for all genes except GAPDH was formulated. To this mixture were added varying ratios of the S-probe for GAPDH mRNA and SA-probes for GAPDH mRNA that were identical to the S-probe, except that they lack the PCR primer binding sequence. The combined concentrations of S- and SA-probes were kept constant. This combination of S- and SA-probes resulted in a proportional attenuation of the GAPDH mRNA signal. One part S-probe combined with nine parts SA-probe is equivalent to 10-fold attenuation. Seven solutions of complete S-probes were formulated to contain the following amounts of GAPDH mRNA attenuation: 667-fold, 334-fold, 133-fold, 67-fold, 50-fold, 33-fold, and 13-fold. The standard CLPA formulation used in these studies contained 67-fold GAPDH mRNA attenuation. These S-probe formulations were used to perform CLPA testing of stabilized blood from two healthy human donors with five technical replicates per condition. Total RNA was isolated from DxCollect blood. To generate templates for each TaqMan assay, approximately 10 ng of total RNA was used to synthesize cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's recommendations. The cDNA were submitted to the University of California, Los Angeles, GenoSeq Core laboratory to obtain TaqMan RT-qPCR expression data by using ABI Life Technologies TaqMan assays performed in triplicate for GAPDH, BAX, PCNA, CDKN1A, and PCNA mRNA with the TaqMan Universal Master Mix II reagent (Life Technologies) on an ABI 7900HT Fast RT-qPCR instrument. Fold change in gene expression was calculated relative to Cq values from GAPDH mRNA. The details of the TaqMan assays used are shown in Table 5.Table 5Details of TaqMan Assays Used to Generate RT-qPCR Data in Figure 7GeneRefSeq ID∗Accession numbers from http://www.ncbi.nlm.nih.gov/nuccore.CLPA target locationCLPA exon/intronTaqMan exon boundaryTaqMan assay locationBAXNM_138761231-371Exon 3/exon 4Exon 3/exon 4306FDXRNM_0244171733-1885Exon12Exon 11/exon 121433GAPDHNM_002046247-384Exon 3/exon 4Exon 3229MRPS18ANM_018135207-349Exon 3/exon 4Exon 3/exon 4291PCNANM_002592852-1010Exon 6/exon 7Exon 5/exon 6819CLPA, chemical ligation-dependent probe amplification; RT-qPCR, quantitative real-time
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