Rapid extraction of RNA and analysis of transcript levels in Chlamydomonas reinhardtii using real-time RT-PCR

magnesium chelatase chlH, chlD and chlI gene expression

Vanessa Lake, Robert D. Willows*

*Corresponding author for this work

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The reverse transcriptase polymerase chain reaction (RT-PCR) is an extremely sensitive technique for detecting RNA transcripts. Real-time RT-PCR using fluorescent dyes and instruments such as the Roche Lightcycler allows real-time kinetic quantification of transcript levels. Here we report a method for the relative quantification of RNA transcripts using real-time RT-PCR that gives results comparable to Northern blotting utilizing 10-100 fold less RNA. We have also optimized a method for the rapid and efficient extraction of RNA from Chlamydomonas reinhardtii. The method is more rapid than other methods tested, allows simultaneous processing of multiple samples, and yields reproducible quantities of total RNA from a fixed number of cells. In addition the purified total RNA is of high quality and polyA-mRNA can be easily isolated. Using these methods we found that the pattern of changes in RNA transcript levels of the magnesium chelatase (Mg-chelatase) genes, chlH, chlD and chlI, of C. reinhardtii grown under synchronous culture conditions in light/dark cycles are similar and that light is involved in this regulation.

Original languageEnglish
Pages (from-to)69-76
Number of pages8
JournalPhotosynthesis Research
Volume77
Issue number1
DOIs
Publication statusPublished - 2003

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