AFLP-based transcript profiling.
This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP-fingerprinting of double-stranded cDNA. The protocol described includes the following steps: the isolation of poly(A)+ RNA from total RNA, the synthesis of double-stranded cDNA, the preparation of template fragments by digestion of the cDNA library with a combination of two restriction enzymes and the ligation of adaptors to the fragment ends, the selective amplification of specific subsets of fragments, and the electrophoretic analysis of these amplification products on standard denaturing polyacrylamide gels. The transcript profiles obtained by this technique are a reliable and efficient tool to identify differentially expressed mRNAs. This unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP-finger his unit presents an alternative to differential display that allows the quantification of transcripts, based on AFLP-finger.
Vos
Pieter
P
Keygene N.V., The Netherlands.
Stanssens
Patrick
P
D054458:Amplified Fragment Length Polymorphism Analysis / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D007202:Indicators and Reagents; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D013698:Templates, Genetic; D014158:Transcription, Genetic
D018076:DNA, Complementary; D007202:Indicators and Reagents; D012333:RNA, Messenger; D004247:DNA; C055927:endodeoxyribonuclease MseI; D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases