Use of terminal transferase-dependent antisense RNA amplification to determine the transcription start site of the Snrpn gene in individual neurons.
We describe here a very sensitive technique for RNA structure analysis and the determination of transcription start sites and demonstrate its use for mapping the start site of the imprinted Snrpn gene in individual hippocampal neurons. The method is adapted from reverse transcription-terminal transferase-dependent PCR (RT-TDPCR) to include amplification of the antisense sequence by in vitro transcription just prior to the final PCR step. The method should be useful for analysis of all genes for which variation in promoter usage and/or differences in RNA secondary structure may be specific to a given cell type or developmental stage.
Buettner
V L
VL
Mammalian Genetics and Molecular Biology Sections, Biology Department, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
LeBon
J M
JM
Gao
C
C
Riggs
A D
AD
Singer-Sam
J
J
D000818:Animals; D004247:DNA; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics*; D014158:Transcription, Genetic
D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA; D004247:DNA; D004253:DNA Nucleotidylexotransferase