Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA

Abstract

Analysis of transcript representation on gene microarrays requires microgram amounts of total RNA or DNA. Without amplification, such amounts are obtainable only from millions of cells. However, it may be desirable to determine transcript representation in few or even single cells in aspiration biopsies, rare population subsets isolated by cell sorting or laser capture, or micromanipulated single cells. Nucleic-acid amplification methods could be used in these cases, but it is difficult to amplify different transcripts in a sample without distorting quantitative relationships between them. Linear isothermal RNA amplification has been used to amplify as little as 10 ng of total cellular RNA, corresponding to the amount obtainable from thousands of cells, while still preserving the original abundance relationships. However, the available procedures require multiple steps, are labor intensive and time consuming, and have not been shown to preserve abundance information from smaller starting amounts. Exponential amplification, on the other hand, is a relatively simple technology, but is generally considered to bias abundance relationships unacceptably. These constraints have placed beyond current reach the secure and routine application of microarray analysis to single or small numbers of cells. Here we describe results obtained with a rapid and highly optimized global reverse transcription#150;PCR (RT-PCR) procedure. Contrary to prevalent expectations, the exponential approach preserves abundance relationships through amplification as high as 3 x 10(11)-fold. Further, it reduces by a million-fold the input amount of RNA needed for microarray analysis, and yields reproducible results from the picogram range of total RNA obtainable from single cells.

The PcG gene Sfmbt2 is paternally expressed in extraembryonic tissues

“…We relied on a PCR based technology for generating quantitative pools of cDNA from very small samples (Iscove et al, 2002). Three different pools of cDNA derived from androgenetic, gynogenetic or biparental blastocysts were labeled and applied to Affymetrix MOE430 A and B expression arrays.…”

“…With this method, the entire population of reverse transcribed cDNAs is transcribed under conditions that quantitatively maintain individual sequence representation. cDNA was submitted to the Microarray Facility of the Ontario Genomics Innovation Centre, Ontario Health Research Institute for labeling and hybridization to Affymetrix MOE 430 A and B chips, using a protocol described in Iscove et al, 2002. Microarray data have been deposited with NCBI GEO, accession number GSE8163. The experiment (Stembase experiment 235) is part of a large collection of microarray experiments aimed at analyzing stem cell function (Perez-Iratxeta, et al, 2005).…”

Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence

“…An alternative to conventional expression profiling methods is now emerging through the development of techniques that allow faithful amplification of mRNA from single cells (21)(22)(23). The cDNA generated can be used as a template to analyze specific genes (22) or can be fragmented and labeled for global expression profiling using DNA arrays (23).…”

Characterization of a method for profiling gene expression in cells recovered from intact human prostate tissue using RNA linear amplification

“…The mean (range) yield of aRNA for PC3 and PC3-M cells was 3.0 mg (1.8-4.0 mg) and 3.1 mg (1.9-4.8 mg), respectively, Table 1. To evaluate the effect of operator experience, aRNA yields from reactions performed early in the 3-month period (reactions 1-6) were compared to those performed late (7)(8)(9)(10)(11)(12), for both PC3 and PC3-M cells. For PC3 cells, mean yields for early and late reactions were 2.9 and 3.0, respectively, whereas for PC3-M cells, they were 3.1 and 3.1, respectively.…”

“…Two different methods can be used to amplify RNA. In the first method, the polymerase chain reaction (PCR) is used to achieve either exponential 9,10 or linear amplification. 11 The second method utilizes in vitro transcription to achieve linear amplification.…”