两种微量RNA扩增方法的比较研究

本研究通过比较体外转录和单引物扩增这两种扩增微量RNA的不同方法,以寻找一种高效的扩增方法。我们用两种不同方法分别扩增小鼠大脑全皮层及第五皮层细胞的RNA,扩增的RNA合成cDNA后进行荧光定量PCR实验,根据PCR结果比较两种不同扩增方法的效率。WT-ovation扩增RNA的效率约为IVT效率的2.8倍;IVT方法扩增后,基因D-C值与引物距离mRNA 3'端的长度及mRNA的长度均存在正线性相关(P<0.05),即引物距离mRNA的3'端越近、mRNA越短,基因D-Ct值越低。而WT-ovation方法扩增后,基因D-Ct值与引物距离mRNA 3'端的长度及mRNA的长度均不存在统计学相关性。与IVT方法相比,WT-ovation方法效率更高,扩增时受影响因素较少、更稳定。     

Advantages of mRNA amplification for microarray analysis

Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been fully validated. Here we demonstrate preserved fidelity in arrays using one or two rounds of mRNA amplification, validated by downstream real-time quantitative PCR. In addition, the quality of the array data was superior to that obtained using total RNA. Based on these results, we recommend routine mRNA amplification for all cDNA microarray-based analysis of gene expression.     

High-fidelity mRNA amplification for gene profiling

The completion of the Human Genome Project has made possible the comprehensive analysis of gene expression, and cDNA microarrays are now being employed for expression analysis in cancer cell lines or excised surgical specimens. However, broader application of cDNA microarrays is limited by the amount of RNA required: 50-200 microg of total RNA (T-RNA) and 2-5 microg poly(A) RNA. To broaden the use of cDNA microarrays, some methods aiming at intensifying fluorescence signal have resulted in modest improvement. Methods devoted to amplifying starting poly(A) RNA or cDNA show promise, in that detection can be increased by orders of magnitude. However, despite the common use of these amplification procedures, no systematic assessment of their limits and biases has been documented. We devised a procedure that optimizes amplification of low-abundance RNA samples by combining antisense RNA (aRNA) amplification with a template-switching effect (Clonetech, Palo Alto, CA). The fidelity of aRNA amplified from 1:10,000 to 1:100,000 of commonly used input RNA was comparable to expression profiles observed with conventional poly(A) RNA- or T-RNA-based arrays.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Ultrasensitive DNA chip: gene expression profile analysis without RNA amplification

We have developed a new DNA chip whose substrate has a unique minute columnar array structure made of plastic. The DNA chip exhibits ultrahigh sensitivity, up to 100-fold higher than that of reference DNA chips, which makes it possible to monitor gene expression profiles even with very small amounts of RNA (0.1-0.01 microg of total RNA) without amplification. Differential expression ratios obtained with the new DNA chip were validated against those obtained with quantitative real-time PCR assays. This novel microarray technology would be a powerful tool for monitoring gene expression profiles, especially for clinical diagnosis.