Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends.
Three assay methods for quantification of the two galactoseoperon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.
Ji
Sang Chun
SC
Department of Biological Science, Chungnam National University, Daejeon, Korea.
Wang
Xun
X
Jeon
Heung Jin
HJ
Yun
Sang Hoon
SH
Lee
Hee Jung
HJ
Lim
Heon M
HM
D020121:5' Untranslated Regions; D017931:DNA Primers / Q000235:genetics; D005690:Galactose / Q000235:genetics; D009876:Operon; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000378:metabolism
D020121:5' Untranslated Regions; D017931:DNA Primers; D012333:RNA, Messenger; D012341:RNA, Ribosomal, 5S; D005690:Galactose