A sensitive technique to clone low abundance receptor transcripts from single microdissected tissue punches.
Tissue microdissection is a rapidly growing technique with wide applicability in the field of gene expression analysis as improved RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) techniques provide the sensitivity to amplify transcription products from increasingly small numbers of cells. In spite of these advances, isolation, cloning and regional localization of rare or low-abundance mRNA from very small tissue samples remain a difficult and challenging task, especially when high degenerate primers are to be used. We have addressed this problem using a combination of optimized techniques and purification steps added between individual reaction steps. The extreme sensitivity resulting from these modifications permits cloning of new members of a closely homologous gene family from only one microdissected tissue sample and widens the applicability of tissue microdissection. Using this protocol, nested degenerate PCR primers were designed to amplify members of the large and relatively homologous olfactory receptor (OR) gene family from RNA extracted from 125-microm diameter punches of tissue microdissected from 16-microm sections of the main olfactory bulb (MOB) of the mouse. Levels of OR mRNA in these punches are extremely low, due to the small volume of tissue and the low abundance of OR mRNA in MOB tissue. Several ORs were amplified, cloned and sequenced from a series of individual tissue punches, and in situ hybridization was used to verify the presence of mRNA corresponding to the cloned OR sequences in MOB sections.
Yang
Xinhai
X
Anesthesia Research Lab, Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA. xinhai,yang@tufts.edu
Marchand
James E
JE
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004210:Dissection / Q000295:instrumentation / Q000379:methods*; D017403:In Situ Hybridization / Q000295:instrumentation / Q000379:methods; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009448:Neurochemistry / Q000295:instrumentation / Q000379:methods*; D009474:Neurons / Q000737:chemistry* / Q000166:cytology; D009830:Olfactory Bulb / Q000737:chemistry* / Q000166:cytology; D018034:Olfactory Receptor Neurons / Q000737:chemistry / Q000648:ultrastructure; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D017729:Presynaptic Terminals / Q000737:chemistry / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D018035:Receptors, Odorant / Q000235:genetics*
D012333:RNA, Messenger; D018035:Receptors, Odorant