Quantification of relative changes in specific mRNAs from frozen whole blood – methodological considerations and clinical implications
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Reidun Øvstebø
Abstract
Background: Based on quantification of relative changes in lipopolysaccharide (LPS)-regulated mRNA transcripts, the present study aimed to establish a robotic method to isolate RNA from stabilized frozen whole blood suitable for gene expression analysis.
Methods: Whole blood (±LPS) was stored in EasyLyse™ solution or PAXgene® tubes (room temperature and −70°C) for comparison of storage methods, then subjected to robotic isolation of total RNA. Yield, quality and relative changes in 11 selected mRNA transcripts were examined. Method precision (% coefficient of variation) for a longitudinal control was established. The influence of globin mRNA from reticulocytes in quantitative RT-PCR was examined.
Results: All storage methods gave a similar high-quality RNA yield. The differences in the 11 specific mRNA quantities stored in EasyLyse or PAXgene® at −70°C were small: mean −0.01 (95% CI –0.19 to 0.17). The CV for mRNAs in the longitudinal control ranged from 3% to 150%. Thus, the number of replicates necessary to detect a 20% difference (power 0.8) ranged from 2–50. Globin mRNA had no influence on quantitative RT-PCR
Conclusions: Based on measuring the relative changes in specific mRNAs in LPS-exposed whole blood, we conclude that PAXgene® tubes stored at −70°C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings.
Clin Chem Lab Med 2007;45:171–6.
References
1. Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA, et al. Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci USA2003;100:1896–901.10.1073/pnas.252784499Search in Google Scholar
2. Debey S, Schoenbeck U, Hellmich M, Gathof BS, Pillai R, Zander T, et al. Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types. Pharmacogenomics J2004;4:193–207.10.1038/sj.tpj.6500240Search in Google Scholar
3. Gibbons GH, Liew CC, Goodarzi MO, Rotter JI, Hsueh WA, Siragy HM, et al. Genetic markers: progress and potential for cardiovascular disease. Circulation2004;109:IV47–58.10.1161/01.CIR.0000133440.86427.26Search in Google Scholar
4. Pahl A. Gene expression profiling using RNA extracted from whole blood: technologies and clinical applications. Expert Rev Mol Diagn2005;5:43–52.10.1586/14737159.5.1.43Search in Google Scholar
5. Radich JP, Mao M, Stepaniants S, Biery M, Castle J, Ward T, et al. Individual-specific variation of gene expression in peripheral blood leukocytes. Genomics2004;83:980–8.10.1016/j.ygeno.2003.12.013Search in Google Scholar
6. Pahl A, Brune K. Stabilization of gene expression profiles in blood after phlebotomy. Clin Chem2002;48:2251–3.10.1093/clinchem/48.12.2251Search in Google Scholar
7. Muirhead KA, Wallace PK, Schmitt TC, Frescatore RL, Franco JA, Horan PK. Methodological considerations for implementation of lymphocyte subset analysis in a clinical reference laboratory. Ann NY Acad Sci1986;468:113–27.10.1111/j.1749-6632.1986.tb42034.xSearch in Google Scholar
8. Stordeur P, Zhou L, Byl B, Brohet F, Burny W, de Groote D, et al. Immune monitoring in whole blood using real-time PCR. J Immunol Methods2003;276:69–77.10.1016/S0022-1759(03)00074-7Search in Google Scholar
9. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet1996;13:399–408.10.1038/ng0896-399Search in Google Scholar PubMed
10. Ovstebo R, Haug KB, Lande K, Kierulf P. PCR-based calibration curves for studies of quantitative gene expression in human monocytes: development and evaluation. Clin Chem2003;49:425–32.10.1373/49.3.425Search in Google Scholar PubMed
11. Goulter AB, Harmer DW, Clark KL. Evaluation of low density array technology for quantitative parallel measurement of multiple genes in human tissue. BMC Genomics2006;7:34.10.1186/1471-2164-7-34Search in Google Scholar
12. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–ΔΔC(T)) method. Methods2001;25:402–8.10.1006/meth.2001.1262Search in Google Scholar
13. Duran EM, Shapshak P, Worley J, Minagar A, Ziegler F, Haliko S, et al. Presenilin-1 detection in brain neurons and FOXP3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase PCR using the ΔΔCt method. Front Biosci2005;10:2955–65.10.2741/1751Search in Google Scholar
14. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet1986;1:307–10.10.1016/S0140-6736(86)90837-8Search in Google Scholar
15. Chai V, Vassilakos A, Lee Y, Wright JA, Young AH. Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples. J Clin Lab Anal2005;19:182–8.10.1002/jcla.20075Search in Google Scholar PubMed PubMed Central
16. Tumor Analysis Best Practices Working Group. Expression profiling – best practices for data generation and interpretation in clinical trials. Nat Rev Genet 2004;5:229–37.10.1038/nrg1297Search in Google Scholar PubMed
17. Shou J, Dotson C, Qian HR, Tao W, Lin C, Lawrence F, et al. Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray. Biomarkers2005;10:310–20.10.1080/13547500500218583Search in Google Scholar PubMed
18. Spijker S, van de Leemput JC, Hoekstra C, Boomsma DI, Smit AB. Profiling gene expression in whole blood samples following an in-vitro challenge. Twin Res2004;7:564–70.10.1375/1369052042663878Search in Google Scholar PubMed
19. Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E, et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res2005;33:e56.10.1093/nar/gni054Search in Google Scholar PubMed PubMed Central
20. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med2006;27:126–39.10.1016/j.mam.2005.12.003Search in Google Scholar PubMed
21. Li D, Butt A, Clarke S, Swaminathana R. Real-time quantitative PCR measurement of thyroglobulin mRNA in peripheral blood of thyroid cancer patients and healthy subjects. Ann NY Acad Sci2004;1022:147–51.10.1196/annals.1318.024Search in Google Scholar
22. Wurfel MM, Park WY, Radella F, Ruzinski J, Sandstrom A, Strout J, et al. Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity. J Immunol2005;175:2570–8.10.4049/jimmunol.175.4.2570Search in Google Scholar
23. Boldrick JC, Alizadeh AA, Diehn M, Dudoit S, Liu CL, Belcher CE, et al. Stereotyped and specific gene expression programs in human innate immune responses to bacteria. Proc Natl Acad Sci USA2002;99:972–7.10.1073/pnas.231625398Search in Google Scholar
24. Fan H, Hegde PS. The transcriptome in blood: challenges and solutions for robust expression profiling. Curr Mol Med2005;5:3–10.10.2174/1566524053152861Search in Google Scholar
25. McPhail S, Goralski TJ. Overcoming challenges of using blood samples with gene expression microarrays to advance patient stratification in clinical trials. Drug Discov Today2005;10:1485–7.10.1016/S1359-6446(05)03644-5Search in Google Scholar
26. Debey 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. Genomics2005;87:653–64.10.1016/j.ygeno.2005.11.010Search in Google Scholar PubMed
©2007 by Walter de Gruyter Berlin New York
Articles in the same Issue
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Articles in the same Issue
- Associations between folate, vitamin B12, homocysteine and pathologies related to aging: the need to consider complex nutrient-nutrient and gene-nutrient interactions and the functional and socio-economic determinants in population-based studies
- Hyperhomocysteinemia and cardiovascular risk in postmenopausal women: the role of folate supplementation
- Association of vitamin B12, folate and homocysteine with functional and pathological characteristics of the elderly in a mountainous village in Sicily
- Association of thyroid dysfunction with vitamin B12, folate and plasma homocysteine levels in the elderly: a population-based study in Sicily
- Desmond (Des) Kenny MSc MCB FICI FRCPath Eur Clin Chem, 1941–2006
- Antibodies to citrullinated peptides: a significant step forward in the early diagnosis of rheumatoid arthritis
- Patient hydration: a major source of laboratory uncertainty
- Determination of the hepatitis C virus subtype: comparison of sequencing and reverse hybridization assays
- Quantification of relative changes in specific mRNAs from frozen whole blood – methodological considerations and clinical implications
- HER-2 protein concentrations in breast cancer cells increase before immunohistochemical and fluorescence in situ hybridization analysis turn positive
- Distinct alleles of mannose-binding lectin (MBL) and surfactant proteins A (SP-A) in patients with chronic cavitary pulmonary aspergillosis and allergic bronchopulmonary aspergillosis
- Novel molecular defect in the platelet ADP receptor P2Y12 of a patient with haemorrhagic diathesis
- Retrospective study of monoclonal gammopathies detected in the clinical laboratory of a Spanish healthcare district: 14-year series
- Renal impairment compromises the use of total homocysteine and methylmalonic acid but not total vitamin B12 and holotranscobalamin in screening for vitamin B12 deficiency in the aged
- Diagnostic value of calculated testosterone indices in the assessment of polycystic ovary syndrome
- Amniotic fibronectin fragmentation and expression of its domains, sialyl and fucosyl glycotopes associated with pregnancy complicated by intrauterine infection
- Predicted influence of sample hematocrit on injected mass of internal standard in mass spectrometry assays utilizing simple protein precipitation for sample preparation
- Evaluation of cardiovascular risk factors and related clinical markers in healthy young Japanese adults
- Cardiac natriuretic peptides after myocardial infarction: relationship with infarct size, left ventricular function and remodelling assessed by 99mTc-sestamibi gated-single photon emission tomography
- Plasma homocysteine cutoff values for venous thrombosis
- Lipid transfer protein activities in subjects with impaired glucose tolerance
- Heat shock protein 70-1 gene expression in pediatric heart surgery using blood cardioplegia
- Biochemical reference intervals for sex hormones with a new AutoDelfia method in aged men
- The Italian External Quality Control Programme for cystic fibrosis molecular diagnosis: 4 years of activity
- Standardization of ischemia-modified albumin testing: adjustment for serum albumin
- Is simultaneous measurement of anti-thyroid peroxidase and anti-thyroglobulin antibodies clinically useful in patients with thyroid dysfunction?
- To the Editor: In reply to Nakano et al. Clin Chem Lab Med 2006;44(5):522–532
- To the Editor: Author reply; Nakano et al. Clin Chem Lab Med 2006;44(5):522–532
- Accreditation of medical laboratories in the European Union
- Prevalence-dependent decision limits for the early detection of type 2 diabetes mellitus in venous blood, venous plasma and capillary blood during glucose challenge
