TY - JOUR
T1 - Rapid extraction of RNA and analysis of transcript levels in Chlamydomonas reinhardtii using real-time RT-PCR
T2 - magnesium chelatase chlH, chlD and chlI gene expression
AU - Lake, Vanessa
AU - Willows, Robert D.
PY - 2003
Y1 - 2003
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0041374293&partnerID=8YFLogxK
U2 - 10.1023/A:1024955916194
DO - 10.1023/A:1024955916194
M3 - Article
C2 - 16228385
AN - SCOPUS:0041374293
SN - 0166-8595
VL - 77
SP - 69
EP - 76
JO - Photosynthesis Research
JF - Photosynthesis Research
IS - 1
ER -