Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme.
BACKGROUND
The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.


RESULTS
Sixty-six Affymetrix microarray slides using lung adenocarcinoma tissue RNAs were analyzed by a statistical re-sampling method in order to detect genes with minimal variation in gene expression. By this approach, we identified DDX5 as a novel internal control for Q-RT-PCR. Twenty-three genes, which were differentially expressed between adjacent normal and tumor samples, were selected and analyzed using 24 paired lung adenocarcinoma samples by Q-RT-PCR using two internal controls, DDX5 and GAPDH. The percentage correlation between Q-RT-PCR and microarray were 70% and 48% by using DDX5 and GAPDH as internal controls, respectively.


CONCLUSIONS
Together, these quantification strategies for Q-RT-PCR data processing procedure, which focused on minimal variation, ought to significantly facilitate internal control evaluation and selection for Q-RT-PCR when corroborating microarray data.
Su
Li-Jen
LJ
Institute of Cancer Research, National Health Research Institutes, Taipei, Taiwan. ljsu@vghtpe.gov.tw <ljsu@vghtpe.gov.tw>
Chang
Ching-Wei
CW
Wu
Yu-Chung
YC
Chen
Kuang-Chi
KC
Lin
Chien-Ju
CJ
Liang
Shu-Ching
SC
Lin
Chi-Hung
CH
Whang-Peng
Jacqueline
J
Hsu
Shih-Lan
SL
Chen
Chen-Hsin
CH
Huang
Chi-Ying F
CY
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000465:Algorithms; D002138:Calibration; D002461:Cell Line, Transformed; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D003627:Data Interpretation, Statistical; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013048:Specimen Handling / Q000379:methods*
C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases