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 5′ 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)(4)(5)(6)(7). Amplifications >105-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 μL; 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 …