Comparison of nucleic acid targets prepared from total RNA or poly(A) RNA for DNA oligonucleotide microarray hybridization.
The aim of this work was to compare DNA microarray results using either total RNA or affinity-purified poly(A) RNA from the same biological sample for target preparation. The high-density oligonucleotide microarrays of both Agilent Technologies (based on two-color detection) and Applied Biosystems (based on single-color detection) were evaluated. Real-time quantitative PCR was used to quantify messenger RNA (mRNA) and ribosomal RNA (rRNA) at different stages of target preparations. Poly(A) RNA versus total RNA target hybridizations exhibited slightly lower correlation coefficients than did self versus self hybridizations (i.e., poly(A) RNA targets vs. poly(A) RNA targets or total RNA targets vs. total RNA targets). Only a small fraction of all transcripts appeared to be significantly over- or underrepresented when total RNA targets or poly(A) RNA targets from the same biological sample were compared. Therefore, the conclusion is that poly(A) affinity purification from total RNA can be omitted during target preparation for routine mRNA expression analysis using high-density oligonucleotide microarrays. Among consistently overrepresented transcripts in total RNA targets were histone mRNAs known to lack poly(A) tails. Therefore, structurally exceptional RNA species can be identified by comparing targets derived from either poly(A) RNA or total RNA using microarray hybridization.
Petersen
Kjell
K
Computational Biology Unit, Bergen Center for Computational Science, University of Bergen, N-5008 Bergen, Norway.
Oyan
Anne Margrete
AM
Rostad
Kari
K
Olsen
Sue
S
Bø
Trond Hellem
TH
Salvesen
Helga B
HB
Gjertsen
Bjørn Tore
BT
Bruserud
Oystein
O
Halvorsen
Ole Johan
OJ
Akslen
Lars Andreas
LA
Steen
Vidar M
VM
Jonassen
Inge
I
Kalland
Karl-Henning
KH
D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D003851:Deoxyribonucleases; D016889:Endometrial Neoplasms / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D008297:Male; D009838:Oligodeoxyribonucleotides; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D011470:Prostatic Hyperplasia / Q000235:genetics; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012335:RNA, Ribosomal; C027903:oligo (dT); D012313:RNA; D003851:Deoxyribonucleases