title: Amplification biases: possible differences among deviating gene expressions.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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 mug 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biologie du Développement et Reproduction UMR 1198; ENVA; CNRS, FRE 2857, Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France. severine.degrelle@jouy.inra.fr
authors.forename: Séverine A
authors.initials: SA
authors.lastname: Degrelle
authors.forename: Christelle
authors.initials: C
authors.lastname: Hennequet-Antier
authors.forename: Hélène
authors.initials: H
authors.lastname: Chiapello
authors.forename: Karine
authors.initials: K
authors.lastname: Piot-Kaminski
authors.forename: Francois
authors.initials: F
authors.lastname: Piumi
authors.forename: Stéphane
authors.initials: S
authors.lastname: Robin
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Renard
authors.forename: Isabelle
authors.initials: I
authors.lastname: Hue
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods