Ideally, gene expression studies by quantitative RT-PCR (qRT-PCR) should be performed using RNA from homogeneous cell populations. Frequently, this means performing qRT-PCR using picogram amounts of total cellular RNA and, in some instances, using total RNA from a single cell.
Technologies such as laser capture microdissection and fluorescence-activated cell sorting make it possible to obtain small cell populations. However, qRT-PCR detection of even high-abundance transcripts from a minute number of cells is challenging, as evidenced by high cycle threshold (CT) values and a lack of consistency between replicates. Often low- and medium-abundance transcripts are not detected at all. Additionally, only a limited number of qRT-PCR reactions can be performed using the total RNA that can be isolated from a small sample.
Epicentre Biotechnologies (www.epibio.com) recently introduced the MessageBooster cDNA Synthesis Kit for qPCR, which enables sensitive and reproducible qRT-PCR of even low-abundance transcripts using total RNA from small cell populations (1 to 50 cells). A MessageBooster reaction amplifies the poly(A) RNA present in a total RNA preparation and then converts the amplified RNA to cDNA, which can be used without purification for qPCR. A MessageBooster reaction preserves the relative transcript abundance of the sample and yields enough cDNA to perform many more qPCR reactions than can be obtained from untreated total RNA samples.
The MessageBooster cDNA Synthesis Kit for qPCR utilizes an improved linear poly(A) RNA amplification process (Figure 1) first described in the laboratory of James Eberwine. Briefly, the poly(A) RNA in a total RNA sample from as little as one cell is converted to double-stranded cDNA.
The cDNA is subsequently used as template in a high-yield T7 in vitro transcription reaction that produces antisense RNA (aRNA, also called cRNA). The aRNA is then reverse transcribed into single-stranded, sense cDNA that can be used without purification for qPCR. A MessageBooster reaction can be completed in one day.