Development of a rapid genetic analysis method using little mRNA from blood samples

在遗传分析中，当mRNA可以从不难获得的组织中获得时，它们是最有益的，因为mRNA既包含基因的核苷酸序列信息，也包含表达水平。但对于只在特定组织中表达的基因来说，很难获得。为了分析仅在难以获得且基因组结构尚未阐明的特定组织中表达的基因的核苷酸序列，我们试图开发一种使用来自血液样本的非特异性mRNA的快速遗传分析方法，从而既不需要基因组DNA结构也不需要基因组DNA结构。从外周血样本中分离RNA后，利用逆转录和聚合酶链反应（RT-PCR）可以扩增出β -肌动蛋白或钠/碘同体（NIS）的cdna，但当使用NIS基因V59E/T354P合并杂合突变的患者血液样本时，与野生型序列相比，V59E突变序列和T354P突变序列扩增效率较低。由于使用0.1微克基因组DNA时，这些等位基因的扩增结果相似，因此，少量cDNA的大量扩增可能会突出单独点突变或与缺少内含子序列的扩增序列变化联合引起的偏扩增。总之，利用血液样本非特异性mRNA的快速遗传分析方法是可行的，但存在偏扩增的瓶颈。

In genetic analysis, mRNA s, most beneficial when they can be obtained from tissues that are not difficult to obtain, as mRNAs contains both the nucleotide sequence information of the gene as well as the expression levels. But for genes that are only expressed in specific tissues that are difficult to obtain., and genomic DNAs are commonly used only when the genomic sturcture has been elucidated To analyze nucleotide sequence of genes that are only expressed in specific tissues that are difficult to obtain and whose genomic sturcture has not been elucidated, we attempted to develop a rapid genetic analysis method using nonspecific mRNA from blood samples so that neither the genomic DNA structure is not needed. After isolating RNA from peripheral blood samples, cDNAs for beta-actin or sodium/iodide symporter(NIS) could be amplified satisfactory using reverse transcription and polymerase chain reaction (RT-PCR), However, when blood Sample of a patient With combined heterozygous mutation of V59E/T354P in the NIS gene was used, V59E mutant sequence and T354P mutant sequence were not amplified efficiently compared to the wild type sequences. As amplification of these alleles were similar when 0.1 microgram genomic DNA was used, tremendous amplification from small amount of cDNA could have made prominent the biased amplification due to point mutation alone or with combination with change in the amplified sequence lacking intron sequence. In conclusion rapid genetic analysis method using nonspecific mRNA from blood samples is workable, but has a bottleneck of biased amplification.