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Optimizing gene-expression analysis for induced sputum

: Holz, Olaf; Hansen, Tanja; Homburg, Ursula; Garn, Holger; Hohlfeld, Jens Michael; Niehof, Monika

American Journal of Respiratory and Critical Care Medicine 189 (2014), Art. A4253
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2014, San Diego/Calif.>
Fraunhofer ITEM ()

Introduction: To enable gene expression analysis in sputum samples of patients with asthma following allergen provocation and treatment with a novel GATA-3 inhibitor ( Identifier: NCT0174376), we compared different RNA protection solutions and RNA isolation procedures and tested five commercially available primers for asthma-related genes (GATA3, TBX21, IL5, IFNG, IL13), as well as primers for two potential reference genes (B2M, CD3E).
Methods: Control sputum of volunteer subjects was collected by a standard induction protocol. Immediately following sputum processing, sputum was conserved in RNAprotect Cell Reagent (Qiagen), which was comparably effective to Trizol (Lifetechnologies), but easier to handle. Due to the limited cell numbers available from sputum samples, RNA was isolated using the RNeasy Micro Kit (Qiagen) including a DNase I digest. RNA quality was checked by Agilent 2100 Bioanalyzer and samples were pooled. After cDNA synthesis (Omniscript, Qiagen), qPCR was performed using a dynamic range of dilutions of cDNA with primers for the above mentioned genes as n=3 technical replicates. qPCR was performed with the ABI 7500® (Agilent/Life Technologies) equipment using a standard program with an annealing temperature of 60°C.
Results: For B2M, CD3E, and GATA3 we found linear expression in sputum samples over a range of at least six log10 concentrations. IFNG, IL5 and IL13 were also detectable, but with lower expression, therefore only 4/4/3 dilution steps could be evaluated. PCR efficiency for all primers (except IL13) amounted to > 85%. TBX21 was only detectable in undiluted control sputum samples but showed sufficient expression in human peripheral blood leukocytes RNA, which was therefore used to control the primer over a wider range of standard dilution steps (PCR efficiency amounted to 90%). Based on these results we recommend the use of at least 0.5 Mio. sputum cells to obtain RNA in sufficient amount and quality.
Conclusion: All primers tested gave a sufficient basal signal in undiluted pooled control sputum samples. Thus, the established analytical system will now be used for samples from asthmatic patients obtained before and after allergen challenge to test if an inflammatory response will be detectable in induced sputum samples.