Development of diagnostics for direct monitoring the pathogenicity of oral bacterium in periodontal pockets

我们开发了一种使用N6-T7引物的原核生物RNA扩增方法，该方法导致经济、高效和线性扩增。使用寡核苷酸微阵列，我们确定了从来自10 μg起始RNA的非扩增cDNA获得的数据的质量和再现性，随后是用于prokayote的Affytek方案，以及扩增的RNA，其分别通过1-循环IVT或2-循环IVT从500 ng或50 ng起始RNA独立地制备。我们的方案始终产生足够的aRNA用于微阵列分析。从使用N6-T7引物的IVT方法获得的数据也与从通过Affyphase方案获得的cDNA获得的结果紧密匹配。另外，在从总RNA合成cDNA的步骤中，通常使用随机引物进行原核转录分析。因此，我们提出的使用N6-T7引物的方法具有以下优点：1）单引物的制备简单且成本低; 2）用于真核生物的基于IVT的商业RNA扩增试剂盒中的大多数试剂可以使用N6-T7引物代替寡聚d [T]_引物用于原核生物<20>; 3）N6-T7引物的扩增方法适用于多种类型的基因探针（寡核苷酸或PCR产物），以及任何标记程序，即单色法（生物素或Cy 5标记）和双色法（Cy 3-和Cy 5-标记）; 4）根据基因探针在阵列上的方向，可以制备有义-aRNA或反义-aRNA; 5）加入两种生物素化试剂可以增强基因的荧光强度，导致用aRNA获得的数据比用未扩增的cDNA获得的数据具有好得多的信噪比。

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.