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

Abstract

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.

Two Components of Aversive Memory inDrosophila, Anesthesia-Sensitive and Anesthesia-Resistant Memory, Require Distinct Domains Within the Rgk1 Small GTPase

“…After total RNA extraction and DNaseI treatment, the RNA quality was assessed with the Experion automated electrophoresis system (Bio-Rad). cDNA was generated from these RNA samples with a Superscript III kit (Invitrogen) and amplified following a previously described protocol (Kurimoto et al, 2007;Tang et al, 2009). The qualities of the final products were determined via qPCR with primers for gapdh, damb, and staufen.…”

“…Rac acts on ASM and the inhibition of Rac activity leads to memory enhancement (Shuai et al, 2010). In addition, both Rac and RGK proteins regulate cytoskeletal remodeling (Luo, 2000;Etienne-Manneville and Hall, 2002). Therefore, we aimed to assess the potential interplay between Rgk1 and Rac by examining whether rgk1 overexpression suppresses the Rac-dependent memory decay.…”

“…ts (McGuire et al, 2004). OK107-Gal4 (Connolly et al, 1996), Df(2R)BSC26 (Parks et al, 2004), and UASrac V12 (Luo et al, 1994). The Gal4 lines 1993-Gal4 and 2765-Gal4 were originally generated and identified as MB-specific drivers in our laboratory (Abe et al, 2014).…”

“…Their roles include the regulation of Ca 2ϩ channel activity (Béguin et al, 2001;Finlin et al, 2003) and the reorganization of cytoskeleton (Pan et al, 2000;Leone et al, 2001;Piddini et al, 2001;Ward et al, 2002). Notably, mammalian REM2 is expressed in the brain (Finlin et al, 2000) and has been shown to be important for synaptogenesis (Ghiretti and Paradis, 2011;Moore et al, 2013), as well as activity-dependent dendritic complexity (Ghiretti and Paradis, 2014;Ghiretti et al, 2014).…”

Transcriptome Sequencing

“…Tang et al (2009) recently published their work on mRNA-seq of single cells using a modified version of a previously published protocol (Kurimoto et al, 2007) to generate full-length cDNA. By utilizing SOLiD sequencing, they acquired and mapped over 100 million reads against the mouse genome and were able to show an increased sensitivity compared to microarray technology.…”