Amplification of Nanogram Amounts of Total RNA by the SMART-Based PCR Method for High-Density Oligonucleotide Microarrays

Abstract

The SMART (switching mechanism at the 5Ј end of RNA templates of reverse transcriptase) method (1, 2 ), in combination with PCR, has been used to amplify minute samples obtained from sources such as laser capture microdissections and biopsies for gene expression analysis using oligonucleotide microarray gene chip technology (3-7 ). Amplifications Ͼ10 5 -fold can be achieved in a short time. However, reports in the literature have rarely discussed the feasibility and reproducibility of PCR-based methods when the amount of starting total RNA in a sample is only a very few nanograms or less, which is often the case for clinical samples.We developed a modified SMART-based PCR protocol with which reliable and robust gene expression data can be obtained from 1 ng of starting total RNA. We also investigated sources of array data variations.The modified protocol (see Supplement 1 in the Data Supplement that accompanies the online version of this Technical Brief at http:www.clinchem.org/content/vol51/ issue12) has three major changes compared with the published standard procedure (5 ): the volume of the reverse transcription reaction is reduced to 5 L; 50 ng of poly(dG/dC) carrier (Sigma) is added to the reverse transcription reaction; and PowerScript reverse transcriptase (BD Biosciences Clontech) is used instead of SuperScript II reverse transcriptase (Invitrogen). When we used the modified protocol to reverse-transcribe and amplify 10, 1, 0.1, and 0.01 ng of human heart or liver total RNA (BD Biosciences Clontech), we obtained ample cDNA from one round of 23, 26, 29, and 32 PCR cycles, respectively (Fig. 1A). The addition of poly(dG/dC) facilitates the reverse transcription of longer transcripts as indicated by larger ranges of cDNA sizes and clearer band patterns, which are important for getting consistent good array data, but it does not adversely interfere with the reactions. Use of Superscript II instead of PowerScript in the reverse transcription step under otherwise similar conditions failed to yield visible amounts of cDNA in the agarose gel when starting total RNA was Յ10 ng, and a second round of PCR had to be performed to yield sufficient cDNA for synthesizing adequate amounts of cRNA for arrays. The number of genes detected on the arrays, however, was considerably lower than that obtained with PowerScript reverse transcriptase.The cDNA obtained with the modified protocol from 1, 10, and 100 ng of human total heart or liver RNA was in vitro-transcribed into complementary RNA (cRNA), which was then labeled with Bio-11-CTP and Bio-16-UTP (Enzo Biochem) by use of MAXIscript TM reagents (Ambion). The cRNA was then hybridized to the human GeneChip H133A (Affymetrix) as described previously (5 ). The numbers of genes detected as present (ϳ30%) were comparable among arrays prepared with various starting amounts of total RNA. We determined the reproducibility of the expression signals detected from these minute samples by running 2 replicate assays. Shown in Fig. 1B are the M-A plots of replicate arrays (M represent...

Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm

“…Other aspects of target preparation can also introduce sources of variation that can affect the correlation between datasets. Reverse transcriptases can vary in their ability to yield useful amounts of cDNA from limiting amounts of RNA [43] and in their ability to generate cDNA that largely maintains the length of the original transcript, an important issue when using oligonucleotide-based microarrays (see later). Comparison of DNA hybridizations (unamplified and PCR), with fragmented aRNA hybridizations (IVT), may also introduce variation due to different hybridization kinetics [44].…”

Single molecule transcription profiling with AFM

“…Genes transcribed at low levels, such as regulatory proteins, exert large biological effects from small changes in expression level [13,18,22]. Considerable work with enzymatic amplification, including PCR-based [23][24][25][26][27][28][29][30][31][32][33], multiple displacement amplification-based (MDA) [24,, and RNA polymerase-based [23,[56][57][58][59][60][61][62][63] protocols, has enabled the use of hybridization microarrays and sequence tag methods to characterize low abundance mRNA samples [64][65][66][67][68][69][70][71][72]. This includes several recent reports of message profiling of single cells [73][74][75][76][77][78][79][80][81][82].…”

“…Single cell transcript profiling studies have found that only large magnitude changes in expression can be quantified for moderate-and low-abundance transcripts [24,27,28,30,35 , 41 , 46 , 57 , 62 ,63,67,83-88]. In several papers, Nygarrd et al [85,88] have argued strongly that fundamental, stochastic effects prevent reliable enzymatic amplification of all species from minute samples; they conclude that high and medium abundance species can be quantitatively amplified, but low copy number species will always be amplified unevenly.…”