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Magnetic quantitative reverse transcription PCR: A high-throughput method for mRNA extraction and quantitative reverse transcription PCR

Jost, Ricarda; Berkowitz, Oliver; Masle, Josette

Description

Over the past few years high-throughput platforms for real-time quantitative PCR have become widely available. The cost of RNA extraction from a large number of samples are, however, quite notable. One method that stands out with respect to free up- or downscaling of sample size and reliability is the isolation of mRNA using oligodeoxythymidylate [oligo(dT)25]-coated magnetic particles. In combining this magnetic separation of mRNA with real-time reverse transcription PCR (RT-PCR), we have...[Show more]

dc.contributor.authorJost, Ricarda
dc.contributor.authorBerkowitz, Oliver
dc.contributor.authorMasle, Josette
dc.date.accessioned2015-12-07T22:14:53Z
dc.identifier.issn0736-6205
dc.identifier.urihttp://hdl.handle.net/1885/17644
dc.description.abstractOver the past few years high-throughput platforms for real-time quantitative PCR have become widely available. The cost of RNA extraction from a large number of samples are, however, quite notable. One method that stands out with respect to free up- or downscaling of sample size and reliability is the isolation of mRNA using oligodeoxythymidylate [oligo(dT)25]-coated magnetic particles. In combining this magnetic separation of mRNA with real-time reverse transcription PCR (RT-PCR), we have achieved a highly reproducible, economic, and fast way of analyzing large sample numbers. One difficulty that has so far prevented the fusion of these techniques relates to accurate mRNA quantification. We present a solution to this problem that enables excellent adjustment of cDNA amounts prior to the real-time PCR. Furthermore, as the mRNA is rapidly isolated from crude plant extracts, our method is widely applicable to herbaceous plant species and various tissue types without cumbersome adjustments. Although designed and tested here for plants, we anticipate that the principles should be applicable to gene expression studies in any other organism. Lastly, due to its flexibility, the method presented here can easily be adapted to specific requirements of various users and has great potential for further automation.
dc.publisherEaton Publishing Co
dc.sourceBioTechniques
dc.subjectKeywords: DNA; Magnetic separation; Plants (botany); RNA; Reverse transcription; Transcription; complementary DNA; messenger RNA; oligodeoxynucleotide derivative; thymidine phosphate; accuracy; Arabidopsis; article; controlled study; gene expression; high throughpu
dc.titleMagnetic quantitative reverse transcription PCR: A high-throughput method for mRNA extraction and quantitative reverse transcription PCR
dc.typeJournal article
local.description.notesImported from ARIES
local.identifier.citationvolume43
dc.date.issued2007
local.identifier.absfor060705 - Plant Physiology
local.identifier.ariespublicationu4070025xPUB2
local.type.statusPublished Version
local.contributor.affiliationJost, Ricarda, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationBerkowitz, Oliver, College of Medicine, Biology and Environment, ANU
local.contributor.affiliationMasle, Josette , College of Medicine, Biology and Environment, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.issue2
local.bibliographicCitation.startpage206
local.bibliographicCitation.lastpage211
local.identifier.doi10.2144/000112534
dc.date.updated2015-12-07T07:36:52Z
local.identifier.scopusID2-s2.0-34547879691
CollectionsANU Research Publications

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