Selective labeling and detection of specific mRNA in a total-RNA sample.
We report a unique approach to selectively label and detect specific RNA using its internal sequence, without separation, reverse transcription, and/or polymerase chain reaction. This approach is especially useful for individual mRNA labeling and detection. To expose mRNA internal sequence for selective labeling and detection, RNase H digestion is used to remove the 3(')-common sequences. We have designed a DNA-2(')-O-Me-RNA hybrid as the template for both RNase H digestion and Klenow extension and have found buffer conditions for both enzymes. Sharing the same template and buffer by these two enzymes largely simplifies experimental procedures. Using this approach, lacZ mRNA was selectively labeled and detected in the presence of thousands of mRNAs. We have successfully demonstrated for the first time the direct labeling and detection of an individual mRNA in a total-RNA sample. The detection sensitivity can reach up to attomole level (5 x 10(-18)mole). This method has great potential in RNA decay and metabolic regulation studies via individual mRNA labeling and in methodology of mRNA direct detection on microchip and direct gene expression profiling, without reverse transcription and polymerase chain reactions.
Huang
Zhen
Z
Department of Chemistry, Brooklyn College and Ph.D Programs of Biochemistry and Chemistry, The CUNY Graduate School, Brooklyn, NY 11210, USA. zhuang@brooklyn.cuny.edu
Alsaidi
Mohammed
M
D001671:Biochemistry / Q000379:methods*; D004256:DNA Polymerase I / Q000737:chemistry; D015342:DNA Probes; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D015342:DNA Probes; D012333:RNA, Messenger; D012313:RNA; D004256:DNA Polymerase I; D016914:Ribonuclease H; D001616:beta-Galactosidase