title: Microarray amplification bias: loss of 30% differentially expressed genes due to long probe - poly(A)-tail distances.
abstract: BACKGROUND
  Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe - poly(A)-tail distances.
  
  
  RESULTS
  Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe - poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe - poly(A)-tail distance >500 nucleotides appearing only after one round of amplification.
  
  
  CONCLUSIONS
  Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA.
authors: Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. a.van.den.berg@path.umcg.nl.
authors: Mirjam C
authors: MC
authors: Boelens
authors: Gerard J
authors: GJ
authors: te Meerman
authors: Johan H
authors: JH
authors: Gibcus
authors: Tjasso
authors: T
authors: Blokzijl
authors: H Marike
authors: HM
authors: Boezen
authors: Wim
authors: W
authors: Timens
authors: Dirkje S
authors: DS
authors: Postma
authors: Harry J M
authors: HJ
authors: Groen
authors: Anke
authors: A
authors: van den Berg
mesh_terms: D016477:Artifacts; D020869:Gene Expression Profiling; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015341:Nucleic Acid Probes / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011061:Poly A / Q000378:metabolism*; D025341:Principal Component Analysis; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012815:Signal Processing, Computer-Assisted; D014407:Tumor Cells, Cultured
chemical_list: D015341:Nucleic Acid Probes; D012333:RNA, Messenger; D011061:Poly A
references: Nat Biotechnol. 2000 Apr;18(4):457-9
references: 10748532
references: Biotechniques. 2006 Feb;40(2):165-6, 168, 170
references: 16526405
references: Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10101-6
references: 10963673
references: Nucleic Acids Res. 2001 Mar 1;29(5):E29
references: 11222780
references: Leukemia. 2002 Apr;16(4):473-7
references: 11960324
references: Biotechniques. 2002 Jun;32(6):1330-4, 1336, 1338, 1340
references: 12074164
references: J Mol Diagn. 2003 Feb;5(1):9-14
references: 12552074
references: Nucleic Acids Res. 2004;32(4):e41
references: 14982960
references: BMC Cancer. 2004 May 19;4:20
references: 15151703
references: Diagn Mol Pathol. 2004 Sep;13(3):151-9
references: 15322426
references: Lancet. 1986 Feb 8;1(8476):307-10
references: 2868172
references: Biometrics. 1989 Mar;45(1):255-68
references: 2720055
references: Proc Natl Acad Sci U S A. 1990 Mar;87(5):1663-7
references: 1689846
references: Science. 1996 Nov 8;274(5289):998-1001
references: 8875945
references: PDA J Pharm Sci Technol. 1998 Mar-Apr;52(2):55-9
references: 9610168
references: Nat Med. 1999 Jan;5(1):117-22
references: 9883850
references: Stat Methods Med Res. 1999 Jun;8(2):135-60
references: 10501650
references: Biotechniques. 2004 Oct;37(4):580, 582-6, 588
references: 15517970
references: Biochem Biophys Res Commun. 2004 Dec 24;325(4):1346-52
references: 15555575
references: BMC Genomics. 2003;4(1):4
references: 12594857
references: Nucleic Acids Res. 2006;34(3):996-1014
references: 16473852
references: Pac Symp Biocomput. 2000;:455-66
references: 10902193