Amplification biases: possible differences among deviating gene expressions
1 Biologie du Développement et Reproduction UMR 1198; ENVA; CNRS, FRE 2857, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France
2 Mathématique, Informatique et Génome UR1077, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France
3 Radiobiologie et Etude du Génome UMR INRA/CEA, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France
4 Mathématiques et Informatique Appliquées UMR INAPG/ENGREF/INRA 518, F-75005 Paris, France
5 Station de Recherches Avicoles, Institut National de la Recherche Agronomique, F-37380 Nouzilly, France
6 Modélisation et Ingénierie des Systèmes Complexes pour le Diagnostic FRE3009 CNRS/BIO-RAD, F-34184 Montpellier Cedex 4, France
7 Biologie des Champignons Filamenteux UFR ESIL, F-13288Marseille Cedex 09, France
BMC Genomics 2008, 9:46 doi:10.1186/1471-2164-9-46Published: 28 January 2008
Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored.
Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3) and somatic tissues (n = 2), we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round). Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number) but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70%) and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID). However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated). In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples.
Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i) the sample used: brain, ovary or embryos, (ii) the enzymatic properties initially inferred (exponential or linear) and (iii) the preliminary optimization of the protocols. Moreover the use of an in-house developed array, small-sized but well suited to the tissues we worked with, was of real interest for the search of differential expressions.