[Optimization of T7-based RNA amplification system for cDNA microarray].
cDNA microarrays are powerful parallel tools for gene expression profiling analysis, which help us to understand the molecular mechanism of diseases and to identify potential targets for therapeutic intervention. However, their broader application are hampered by the large amount of RNA required: up to 200 microg of total RNA or 5 microg of mRNA for one chip, making analysis of small samples difficult. In this work, combined with a template switching effect, the T7 RNA linear amplification procedure was optimized, providing multiple copies of anti-sense RNA with reduced sample inputs: no more than 3 microg total RNA for one chip. Using the same RNA sample in all cases, the anti-sense RNA labeling method was compared with standard total RNA and mRNA methods by two sets of self-comparison experiments. Furthermore, the three methods in profiling analysis were compared with the same pair RNA samples. All the results indicated that the fidelity, reproducibility, and reliability showed no significant difference with conventional total RNA or mRNA microarrays.
Li
Tao
T
Shanghai BioStar Genecip, Inc., Shanghai.
Li
Yao
Y
Han
Zhi-Yong
ZY
Chen
Qin
Q
Qiu
Min-Yan
MY
Ni
Sheng
S
Jiang
Zhen-Ling
ZL
Xie
Yi
Y
Mao
Yu-Min
YM
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D020869:Gene Expression Profiling; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D014764:Viral Proteins
D012333:RNA, Messenger; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases