Real-time quantitative RT-PCR method for estimation of mRNA level of CCAAT/enhancer binding protein in the central nervous system of Lymnaea stagnalis.
The fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify mRNA level in cells and tissues and is now a crucial tool for basic biological researches and biotechnology. In the present study, on the basis of the real-time quantitative RT-RCR, we detected and quantified mRNA copies of the transcription factor, CCAAT/enhancer binding protein (C/EBP: an immediate-early gene that is involved in synaptic plasticity and learning and memory) in the central nervous system of the pond snail Lymnaea stagnalis. We designed the primer set and the probe in the specific insert for the detection of Lymnaea C/EBP (LymC/EBP) clone 1. This insert is not contained in LymC/EBP clone 2 by alternative splicing. The copy number of LymC/EBP clone 1 was linearly decreased relative to the dilution of cDNA, and it was estimated 30 copies/microl in test sample. The availability of the present study showed that the real-time quantitative RT-PCR technique is more accurate and more specific for the detection and quantification of the mRNA level of genes in L. stagnalis than the other PCR methods.
Hatakeyama
D
D
Division of Biological Sciences, Graduate School of Science, Creative Research Initiative Sousei, Hokkaido University, Sapporo 060-0810, Japan. dai@sci.hokudai.ac.jp
Sadamoto
Hisayo
H
Ito
E
E
D017398:Alternative Splicing; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D002490:Central Nervous System / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D008195:Lymnaea / Q000378:metabolism*; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012984:Software
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA