The quantitative distribution of a putative PKC epsilon mRNA in Limulus central nervous system by modified competitive RT-PCR.
Recently, a full length cDNA for the epsilon (epsilon) isoform of protein kinase C (PKC) was cloned and sequenced from a cDNA library for the horseshoe crab, Limulus polyphemus. This multifunctional enzyme has been implicated in the modulation of the choline cotransporter in Limulus and the epsilon isoform has been identified in homogenates from its central nervous system (CNS). RT-PCR has proven to be a very useful method for quantifying even a few molecules of mRNA in tissue samples. A modified competitive RT-PCR was used here to quantify a putative PKC epsilon mRNA in Limulus CNS preparations. First, we replaced normally used oligo dT and random primers generated from mRNA with a PKC epsilon specific (3' end) primer P4. Then we used modified nucleotides to extend sample life in storage and finally, we used only annealing and denaturing temperatures during PCR to reduce background. The modified method was used for the first time to quantify PKC epsilon mRNA from three distinct areas of the CNS in Limulus. Results revealed high levels of PKC epsilon mRNA in the corpora pedunculata, in the abdominal ganglia and in the brain ring. These results indicate that PKC epsilon mRNA is broadly distributed throughout the Limulus CNS. Importantly, this modified competitive RT-PCR technique was successfully applied to the quantitation of specific mRNA from Limulus nervous tissue for which no internal standard is available commercially.
Cao
Z
Z
Department of Anatomy and Physiology, Center for Molecular and Behavioral Neuroscience, School of Medicine, Meharry Medical College, 1005 Dr. D. B. Todd Boulevard, Nashville, TN 37208-3599, USA.
Wang
Y
Y
Reid
E A
EA
McShepard
G
G
Kemp
M
M
Newkirk
R F
RF
Townsel
J G
JG
D000818:Animals; D015152:Blotting, Northern; D002490:Central Nervous System / Q000737:chemistry*; D005260:Female; D006737:Horseshoe Crabs / Q000033:anatomy & histology / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics*; D008297:Male; D011493:Protein Kinase C / Q000235:genetics*; D051744:Protein Kinase C-epsilon; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
D007527:Isoenzymes; D012333:RNA, Messenger; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon