Amplification of RNA transcripts using terminal continuation.
A new methodology has been developed to amplify RNA from minute amounts of starting material. Specifically, an efficient means of second-strand (ss) cDNA synthesis using a sequence-specific 'terminal continuation' (TC) method is demonstrated. An RNA synthesis promoter is attached to the 3' and/or 5' region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications including gene expression profiling, cDNA microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translations and downstream proteomic applications. The TC RNA amplification methodology offers high sensitivity, flexibility, and throughput capabilities. A likely mechanism is that the TC primer binds preferentially to GC-rich CpG islands flanking 5' regions of DNA that contain promoter sequences. Following TC RNA amplification, a large proportion of genes can be assessed quantitatively as evidenced by bioanalysis and cDNA microarray analysis in mouse and human postmortem brain tissues.
Che
Shaoli
S
Center for Dementia Research, Nathan Kline Institute, New York University School of Medicine, Orangeburg, NY 10962, USA.
Ginsberg
Stephen D
SD
D000818:Animals; D001921:Brain / Q000166:cytology / Q000502:physiology; D001923:Brain Chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA