Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.
Anal Biochem
; 321(2): 244-51, 2003 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-14511690
Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Análise de Sequência com Séries de Oligonucleotídeos
/
Perfilação da Expressão Gênica
/
Técnicas de Amplificação de Ácido Nucleico
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2003
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Estados Unidos