Scaling up in vitro transcription synthesis of RNA standards for competitive quantitative RT-PCR: Looking for bigger yields

Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the st...

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Bibliographic Details
Published in:Analytical biochemistry 2009-02, Vol.385 (1), p.179-181
Main Authors: Gonzalez-Perez, Idania, Rosa, Iria García de la, Cayarga, Anny Armas, Hernández, Yenitse Perea, González, Yaimé Josefina González, Víctores, Yoamna Rosario, Dueñas-Carrera, Santiago
Format: Article
Language:English
Subjects:
Hepacivirus - genetics
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction - methods
Reverse Transcriptase Polymerase Chain Reaction - standards
RNA, Viral - biosynthesis
RNA, Viral - genetics
Transcription, Genetic
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Summary:Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the standard Riboprobe kit (1 μg RNA/μg template, 1×) and the optimized variant for high yields (5–10×). They were also as good as the ones described for the high RNA production kit RiboMAX (10–20×), exceeding them in approximately 70% of reactions. Our best results, achieved in a 500-μl format, were three to five times higher than the ones reported for the Promega kit.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2008.10.009