Journal
LABORATORY INVESTIGATION
Volume 84, Issue 1, Pages 131-137Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/labinvest.3700005
Keywords
mRNA; cDNA microarray; brain; expression profiling; in vitro transcription; amplified RNA; molecular fingerprint; single-cell microdissection
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Funding
- NATIONAL CANCER INSTITUTE [R21CA094520] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R01NS043939] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [R01AG043375] Funding Source: NIH RePORTER
- NCI NIH HHS [CA94520] Funding Source: Medline
- NIA NIH HHS [R01 AG043375, P01 AG014449] Funding Source: Medline
- NINDS NIH HHS [NS43939] Funding Source: Medline
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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 30 and/or 50 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 50 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.
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