近年來,,在小RNA的研究方面,長非編碼RNA(long noncoding RNA, lncRNA)研究成為熱點,。
lncRNA是一類轉(zhuǎn)錄本長度超過200個核苷酸的RNA分子的總稱,。lncRNA的表達水平相對于編碼蛋白的基因一般比較低,。多數(shù)lncRNA雖然不直接參與基因編碼和蛋白質(zhì)合成,但在基因轉(zhuǎn)錄后調(diào)控,、剪切和修飾具有十分重要的功能,,也在很多生命活動中均起著舉足輕重的作用。與疾病的發(fā)生發(fā)展,、診斷治療密切相關(guān),,迅速成為當今分子生物學(xué)最熱門的前沿研究領(lǐng)域之一。另外,,lncRNA的亞細胞位置上也呈多樣化,,在細胞核、細胞質(zhì)和細胞器均有分布,,甚至某些lncRNA具有獨特的亞細胞位置,,有可能是全新的亞細胞構(gòu)成。
雖然研究甚少,,但人們對lncRNA研究正在逐步加深。比如,,文獻中通過絕對定量的方式,,可以對lncRNA分子進行直接定量,從而大致判斷其對基因表達的調(diào)控屬于順式還是反式作用(具體見全文文獻Figure 3和Figure 4),;對不同lncRNA以及管家基因在亞細胞水平上的表達做了分析(具體見全文文獻Figure 3,、Figure 4和Figure 5);
值得一提的是,,該實驗室對ddPCR在lncRNA研究方面相對于qPCR技術(shù)發(fā)表了他們的看法,,非常具有推廣和借鑒意義。
總之,,lncRNA的異常和疾病關(guān)系密切,,將成為理解疾病、尋找疾病分子標記物,、藥物靶點的新的研究方向,。(生物谷Bioon.com)
生物谷推薦英文全文下載:Digital Quantitation of Potential Therapeutic Target RNAs
NUCLEIC ACID THERAPEUTICS DOI: 10.1089/nat.2013.0427
Accurate determination of the amount of a given RNA within a cell is necessary to gain a full understanding of the RNA’s function and regulation. Typically, the abundance of RNA is measured by quantitative polymerase chain reaction (qPCR). With qPCR, however, absolute quantification is not possible unless an adequate reference standard curve is generated. The method is not well suited for detecting low copy number templates and values vary depending on the specific primers used. To overcome these drawbacks, digital PCR (dPCR) has been developed to obtain exact values for RNA copies in a sample. Here we report the characterization of droplet digital PCR (ddPCR). We used ddPCR to quantify long noncoding RNAs from various subcellular compartments within human cells and found that results obtained using ddPCR parallel those from qPCR. Mutant huntingtin (HTT) protein is the cause of Huntington’s Disease, and we show that we can quantify human HTT messenger RNA and discriminate between the mutant and wild-type HTT alleles using ddPCR. These results
reveal insights into the design of experiments using ddPCR and show that ddPCR can be a robust tool for identifying the number of RNA species inside of cells.