New data on robustness of gene expression signatures in leukemia: comparison of three distinct total RNA preparation procedures.
BACKGROUND
Microarray gene expression (MAGE) signatures allow insights into the transcriptional processes of leukemias and may evolve as a molecular diagnostic test. Introduction of MAGE into clinical practice of leukemia diagnosis will require comprehensive assessment of variation due to the methodologies. Here we systematically assessed the impact of three different total RNA isolation procedures on variation in expression data: method A: lysis of mononuclear cells, followed by lysate homogenization and RNA extraction; method B: organic solvent based RNA isolation, and method C: organic solvent based RNA isolation followed by purification.


RESULTS
We analyzed 27 pediatric acute leukemias representing nine distinct subtypes and show that method A yields better RNA quality, was associated with more differentially expressed genes between leukemia subtypes, demonstrated the lowest degree of variation between experiments, was more reproducible, and was characterized with a higher precision in technical replicates. Unsupervised and supervised analyses grouped leukemias according to lineage and clinical features in all three methods, thus underlining the robustness of MAGE to identify leukemia specific signatures.


CONCLUSIONS
The signatures in the different subtypes of leukemias, regardless of the different extraction methods used, account for the biggest source of variation in the data. Lysis of mononuclear cells, followed by lysate homogenization and RNA extraction represents the optimum method for robust gene expression data and is thus recommended for obtaining robust classification results in microarray studies in acute leukemias.
Campo Dell'Orto
Marta
M
University of Padua, Laboratory of Molecular Diagnostic, Department of Pediatric Oncology, Padua, Italy. marta.campodellorto@unipd.it
Zangrando
Andrea
A
Trentin
Luca
L
Li
Rui
R
Liu
Wei-min
WM
te Kronnie
Geertruy
G
Basso
Giuseppe
G
Kohlmann
Alexander
A
D000704:Analysis of Variance; D014408:Biomarkers, Tumor / Q000235:genetics; D020869:Gene Expression Profiling; D015973:Gene Expression Regulation, Leukemic; D005821:Genetic Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007938:Leukemia / Q000235:genetics* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000378:metabolism; D012334:RNA, Neoplasm; D012997:Solvents / Q000737:chemistry
D014408:Biomarkers, Tumor; D012334:RNA, Neoplasm; D012997:Solvents; D012313:RNA