Microarray amplification bias: loss of 30% differentially expressed genes due to long probe - poly(A)-tail distances.
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.
Boelens
Mirjam C
MC
Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. a.van.den.berg@path.umcg.nl.
te Meerman
Gerard J
GJ
Gibcus
Johan H
JH
Blokzijl
Tjasso
T
Boezen
H Marike
HM
Timens
Wim
W
Postma
Dirkje S
DS
Groen
Harry J M
HJ
van den Berg
Anke
A
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
D015341:Nucleic Acid Probes; D012333:RNA, Messenger; D011061:Poly A
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