Press Release, EPICENTRE® Biotechnologies

|Press Release|

Perform a MessageBOOSTER™ cDNA Synthesis Kit for qPCR reaction directly from a whole-cell lysate.

The MessageBOOSTER™ cDNA Synthesis Kit for qPCR greatly improves the sensitivity and reproducibility of qRT-PCR for low-, medium- and high-abundance transcripts from as little as 1 cell.^1,2 A MessageBOOSTER reaction utilizes a linear RNA amplification process that produces large amounts of anti-sense RNA (aRNA or cRNA) from a total RNA sample and then converts the aRNA to single-stranded cDNA that is ready for qPCR (Figure 1). A MessageBOOSTER reaction preserves the relative transcript abundance (gene expression profile) of the sample³ and greatly increases the number of RT-PCR reactions that can be performed from very small samples of cells¹.

Since the introduction of the MessageBOOSTER kit, we have frequently been asked if a MessageBOOSTER reaction can be used to produce cDNA directly from whole-cell lysates without the need for first purifying the total cellular RNA. In a study to be published in the EPICENTRE Forum newsletter v.13 no.3 (in press) we demonstrate that a MessageBOOSTER reaction efficiently produces cDNA directly from cell lysates of 1 and 10 cells and that the cDNA produced enables sensitive qPCR detection of a medium- and low-abundance transcript.

Figure 1. A MessageBOOSTER™ reaction utilizes a linear RNA amplification process to amplify the poly(A) RNA (mRNA) from total RNA of 1 to 50 cells (10 pg to 500 pg total RNA) and then coverts the aRNA produced to cDNA that is ready for qPCR.

Methods

Cell capture: MessageBOOSTER reaction performed directly using whole-cell lysates was demonstrated using cultured HeLa cells. Trypsinized HeLa cells were pelleted, washed and then resuspended in 1X PBS. The number of cells/ml was measured using a cytometer. Ten-cell samples were collected by serial dilution and the number of cells captured was verified using an inverted microscope.

Single-cell samples were obtained by trapping approximately one HeLa cell in a capillary (Wiretrol^®, Drummond Scientific). The trapped cell was spun down into a microcentrifuge tube at 3500 rpm for 2 minutes.

Cell lysis and inhibition of cellular RNases: There are many procedures for generating whole-cell lysates from eukaryotic cells. We chose a simple freeze/thaw process. The collected cell(s) were frozen and then thawed at room temperature. Ten Units of ScriptGuard™ RNase Inhibitor (EPICENTRE) was added to each sample lysate.

Figure 2. cDNA produced by a MessageBOOSTER™ cDNA Synthesis Kit for qPCR reaction directly from a 10-cell lysate greatly improves qPCR results. qPCR detection of B2M transcript using:

_____	cDNA produced using the MessageBOOSTER kit from a 10-cell lysate and diluted 1:50 prior to qPCR.
_____	cDNA produced using the MessageBOOSTER kit from 100 pg of purified total RNA and diluted 1:50 prior to qPCR.
_____	cDNA produced from a 10-cell lysate without benefit of a MessageBOOSTER reaction and used undiluted for qPCR.
_____	cDNA produced from 100 pg of purified total RNA, without benefit of a MessageBOOSTER reaction and used undiluted for qPCR.
_____	No-template control.

Removal of genomic DNA: Since the MessageBOOSTER reaction incorporates a DNase treatment step, there is no need to treat the cell lysates to remove genomic DNA. A mock MessageBOOSTER reaction performed without the first reverse transcription step, and thus no cDNA synthesis, produced no discernable signal after 45 PCR cycles (no-template control).

References

Grunenwald, H. et al. (2006) EPICENTRE Forum 13(1), 7.
DeFazio, R.A., et. al. (2006) J. Neurosci. 26(15), 3971.
Meis, J.E. (2006) EPICENTRE Forum 13(2), 4.