Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs.
Plant Signal Behav
; 8(8)2013 Aug.
Article
em En
| MEDLINE
| ID: mdl-23673353
ABSTRACT
We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.
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Base de dados:
MEDLINE
Assunto principal:
RNA Nuclear Pequeno
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DNA Complementar
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
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MicroRNAs
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Conformação de Ácido Nucleico
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article