An improved primer extension method for detection of mRNA start-points using non-radioactive digoxigenin-labeling primers.
An improved primer extension method using non-radioactive digoxigenin (DIG)-labeling primers is described which uses a commercially-available DIG-labeling and detection system to perform alternative hybridization-based "labeling" procedure of DNA markers and DIG chemiluminescent detection assay. The time-consuming annealing step and relatively low specificity of conventional protocol are also improved considerably by an application of one-step primer/mRNA annealing procedure and subsequent high-temperature reverse transcription reaction. This new protocol is convenient, simple, cost-effective and safe, and can allow the detection of even low abundance mRNA start-points in enriched poly(A)(+) RNA samples.
Qi
Xiaoting
X
College of Life Science, Capital Normal University, Beijing, PR China. qixiaoting@mail.cnu.edu.cn
Chai
Xiaoqing
X
Chai
Tuanyao
T
D017931:DNA Primers / Q000378:metabolism*; D004076:Digoxigenin / Q000378:metabolism*; D027805:Phaseolus / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D014026:Nicotiana / Q000378:metabolism; D024363:Transcription Initiation Site
D017931:DNA Primers; D012333:RNA, Messenger; D004076:Digoxigenin