2006 Fiscal Year Final Research Report Summary
Development of diagnostics for direct monitoring the pathogenicity of oral bacterium in periodontal pockets
| Project/Area Number |
16591867
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | Nihon University |
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Principal Investigator |
HIRATSUKA Koichi Nihon University, School of Dentistry at Matsudo, Lecturer(Full-Time), 松戸歯学部, 講師 (80246917)
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| Project Period (FY) |
2004 – 2006
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| Keywords | RNA amplification / primer / in vitro transcription / prokaryote / microarray / gene expression |
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| Research Abstract |
We developed an RNA amplification method for prokaryotes using a N6-T7 primer, which led to an economical, efficient, and linear amplification. Using an oligonucleotide microarray, we determined the quality and the reproducibility of the data obtained from the non-amplified cDNA from 10 μg of starting RNA, followed by an Affymetrix protocol for prokayote, and amplified RNAs, which were independently prepared from 500 ng or 50 ng of starting RNA by 1-cycle IVT or 2-cycle IVT, respectively. Our protocol consistently produced sufficient aRNA for microarray analysis. The data obtained from the IVT methods using the N6-T7 primer also closely matched the results from the cDNA obtained by the Affymetrix protocol.
The linear RNA amplification by IVT is well-known as a standard technique in eukaryotes. In addition, a random primer is generally used at the step of cDNA synthesis from total RNA for prokaryotic transcription analysis. Therefore, our proposal procedure using the N6-T7 primer has the following advantages : 1) the manufacturing of the single primer is simple and low-cost ; 2) most of the reagents in commercial IVT-based RNA amplification kits for eukaryotes can be utilized for prokaryotes using the N6-T7 primer instead of the oligo d [T]_<20> primer ; 3) our amplification procedure using the N6-T7 primer can be adapted for many types of gene probes (oligonucleotide or PCR products) on microarrays, and for any labeling procedures, i.e. one-color method (biotin-or Cy5-labeling) and two-color method (Cy3-and Cy5-labeling) ; 4) sense-aRNA or antisense-aRNA can be prepared depending on the orientation of the gene probes on the arrays ; 5) fluorescent intensities of genes can be enhanced by adding two kinds of biotinylated reagents in comparison to the end-labeled cDNA recommended by an Affymetrix protocol, resulting in the data obtained with aRNA having a much better signal-to-noise ratio than that with non-amplified cDNA.
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