Single primer amplification (SPA) of cDNA for microarray expression analysis

Abstract

The potential of expression analysis using cDNA microarrays to address complex problems in a wide variety of biological contexts is now being realised. A limiting factor in such analyses is often the amount of RNA required, usually tens of micrograms. To address this problem researchers have turned to methods of improving detection sensitivity, either through increasing fluorescent signal output per mRNA molecule or increasing the amount of target available for labelling by use of an amplification procedure. We present a novel DNA-based method in which an oligonucleotide is incorporated into the 3' end of cDNA during second-strand cDNA synthesis. This sequence provides an annealing site for a single complementary heel primer that directs Taq DNA polymerase amplification of cDNA following multiple cycles of denaturation, annealing and extension. The utility of this technique for transcriptome-wide screening of relative expression levels was compared to two alternative methodologies for production of labelled cDNA target, namely incorporation of fluorescent nucleotides by reverse transcriptase or the Klenow fragment. Labelled targets from two distinct mouse tissues, adult liver and kidney, were compared by hybridisation to a set of cDNA microarrays containing 6500 mouse cDNA probes. Here we demonstrate, through a dilution series of cDNA derived from 10 micro g of total RNA, that it is possible to produce datasets comparable to those produced with unamplified targets with the equivalent of 30 ng of total RNA. The utility of this technique for microarray analysis in cases where sample is limited is discussed.

Candidate testis‐determining gene, Maestro (Mro), encodes a novel HEAT repeat protein

“…To overcome the limitations imposed by the small amounts of RNA available from embryonic tissue, a Taq polymerasebased amplification procedure was used before labeling of RNA (see Experimental Procedures section and Smith et al, 2003). This procedure allowed hybridizations to be performed with as little as 50 ng of total RNA without significant loss of the ability to reliably detect differential expression.…”

“…Total RNA was prepared from embryonic gonads at 13.5 dpc as previously described (Grimmond et al, 2000). Due to the limiting amounts of RNA available, we used an amplification strategy based on PCR (for details, see Smith et al, 2003). Briefly, first-strand cDNA was generated by using a modified oligo-dT primer (Van Gelder et al, 1990).…”

Sensitive Gene Expression Profiling of Human T Cell Subsets Reveals Parallel Post-Thymic Differentiation for CD4+ and CD8+ Lineages

“…However self-on-self experiments do not allow addressing the reproducibility of differential gene expression. Therefore, following a more rigorous approach, a set of microarrays (duplicates, 12 microarrays) was obtained with amplified material from 1, 10, 100, 1000, or 10000 Jurkat T cells hybridized against the same numbers of Raji B-cells (all titrated by dilution) and compared with profiles obtained with nonamplified material from 10 7 Jurkat cells vs Raji cells. Correlation coefficients close to 0.9 were obtained between the nonamplified material and the amplified material down to 100 Jurkat vs 100 Raji cell samples (Fig.…”

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.…”

“…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. 12,13 With amplification of a large population of genes, distortion of initial relationships becomes an important consideration.…”