Improved measurement of absolute mRNA quantity without reverse transcription.
Anal Biochem
; 579: 1-8, 2019 08 15.
Article
em En
| MEDLINE
| ID: mdl-31078491
ABSTRACT
Gene expression studies using microarrays have provided important insights into understanding the mechanisms of transcriptional regulation in a variety of biological and disease phenomena. In a previous study, we developed Photo-DEAN, a universal-microarray-based RNA quantification method that enabled reverse transcription-free multiplex measurement of the absolute amount of RNA. Photo-DEAN promotes high-throughput and bias-less transcriptome analysis without the need for common controls or additional complicated normalization steps. In this study, we empirically identified two conditions (individual specificity and uniform duplex stability) necessary for in silico design of probe sequences, allowing the Photo-DEAN method to accurately measure the absolute amount of target RNA in total RNA. We then demonstrated that using the modified probe design conditions, the Photo-DEAN method successfully measured the absolute amount of pgi mRNA spiked into E. coli total RNA. The measurement was performed at five different sites in the coding region of pgi mRNA, exhibiting no significant site dependence. Theoretical considerations suggested that probe sequences longer than the previously used 30-bases better satisfy the necessary design conditions.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
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Perfilação da Expressão Gênica
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Transcrição Reversa
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Glucose-6-Fosfato Isomerase
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article