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.     

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.     

cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification.

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.     

Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis

We describe here a protocol for the representative amplification of global mRNAs from typical single mammalian cells to provide a template for high-density oligonucleotide microarray analysis. A single cell is lysed in a tube without purification and first-strand cDNAs are synthesized using a poly(dT)-tailed primer. Unreacted primer is specifically eliminated by exonuclease treatment and second strands are generated with a second poly(dT)-tailed primer after poly(dA) tailing of the first-strand cDNAs. The cDNAs are split into four tubes, which are independently directionally amplified by PCR, and then recombined. The amplified products (approximately 100 ng) show superior representation and reproducibility of original gene expression, especially for genes expressed in more than 20 copies per cell, compared with those obtained by a conventional PCR protocol, and can effectively be used for quantitative PCR and EST analyses. The cDNAs are then subjected to another PCR amplification with primers bearing the T7 promoter sequence. The resultant cDNA products are gel purified, amplified by one final cycle and used for isothermal linear amplification by T7 RNA polymerase to synthesize cRNAs for microarray hybridization. This protocol yields cDNA templates sufficient for more than 80 microarray hybridizations from a single cell, and can be completed in 5-6 days.     

Detection of biomarker gene expression by real-time polymerase chain reaction using amplified ribonucleic acids from formalin-fixed random periareolar fine needle aspirates of human breast tissue

OBJECTIVE: To address the detection of breast cancer biomarker gene expression in formalin-fixed random periareolar fine needle aspiration (RPFNA) samples of benign breast tissue collected during breast cancer prevention trials by quantitative real-time polymerase chain reaction (qPCR). STUDY DESIGN: Formalin-fixed breast epithelial cells collected by RPFNA and processed as thin layer preparations were isolated by laser capture microdissection (LCM). Ribonucleic acid (RNA) was extracted and amplified using a single round of T7-based linear amplification followed by quality assessment and biomarker assay using TaqMan chemistry. RESULTS: More than 80% of RPFNA samples yielded RNA of sufficient quantity and quality for measurement of a panel of biomarker genes following a single round of linear amplification. RNA and protein expression for estrogen receptor alpha, as assessed by LCM/qPCR and immunohistochemistry, were correlated. Amplification plots were similar for cDNA standards and cDNA derived from RPFNA samples. CONCLUSION: Assessment of gene expression using amplified RNA from microdissected formalin-fixed RPFNAs can increase the number of biomarkers used during breast cancer chemoprevention trials.     

一种用于基因芯片可靠的双轮RNA线性扩增技术

DNA微阵列能在一次实验中检测成千上万个基因的表达情况, 有助于阐明疾病发生的分子机制及发现新的诊治靶标.但常规方法需要大量RNA, 因而基于T7 RNA线性扩增技术逐渐成为微阵列表达谱实验中最常用的探针制备方法.本方法将实验步骤进一步改进,增加额外的一轮体外转录,并结合Klenow酶标记技术来制备cDNA靶标和寡核苷酸芯片杂交.从纳克量级的总RNA起始,本方法和常规的RNA单轮线性扩增法相比,仍然准确地保留了约70%的基因表达信息.同一RNA样本的自身比较实验及重复实验结果也显示,该方法具有较高的可靠性和重复性.RNA双轮体外扩增法需要的起始RNA相对于常规的单轮扩增法减少了很多（10 ng甚至更少）,因而非常适合分析那些只能提供微量RNA的样本.     

Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 × 10⁵ cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K_m) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.