Microanalysis of gene expression in tissues using T7-SAGE: serial analysis of gene expression after high-fidelity T7-based RNA amplification.
In this unit, the authors describe a new technique, T7-SAGE, in which a high-fidelity T7 amplification step is combined with SAGE analysis. In order to avoid extra PCR or other forms of amplification, the authors incorporate only two cycles of T7-based RNA amplification as the initial step. This T7-based amplification step has high accuracy. In addition, T7 RNA polymerase has high processivity and functions effectively even when broad stretches of nucleotides are being amplified. Although no protocol that includes an amplification step can claim to permit determination of absolute transcript number, since slight changes in estimated transcript frequency are always possible, T7 procedures appear to be the safest to date. This new T7-SAGE procedure should facilitate application of SAGE for gene-expression profiling using minimal quantities of starting material, such as from embryonic tissues and microdissected cells from histological sections of tissues.
Sakai
Takayoshi
T
National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
Larsen
Melinda
M
Yamada
Kenneth M
KM
D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000737:chemistry*
D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases