MessageBOOSTER™ cDNA Synthesis Kit for qPCR
Produce amplified amounts of cDNA from precious, limiting total RNA samples without introducing bias.
- Perform RNA amplification on purified total RNA followed by cDNA synthesis and detection
- Amplify with this high-fidelity, linear RNA amplification process that preserves the relative transcript abundance of the sample.
- Get more data out of precious samples – use less RNA for more RT-qPCR reactions
- Readily and reproducibly detect even low-abundance transcripts in RNA from a single cell (CT values <35 cycles)
- Collect small RNA samples less often.
- Fast protocol amplifies and synthesizes cDNA in only 1 day.
- Significantly increasing the number of qRT-PCRs that can be obtained from very small amounts of total RNA, as little as one cell (10 pg).
- Generation of large amounts of cDNA from very small samples of intact total RNA for archival purposes.
The MessageBOOSTER™ cDNA Synthesis Kit for qPCR enables the user to perform sensitive qPCR amplifications using the total RNA from very small populations of cells, even from as little as one cell (Table 1).
The kit amplifies the mRNA contained in a small total RNA sample and then converts the amplified RNA to cDNA that is ready for PCR or qPCR (Fig. 1). The MessageBOOSTER cDNA Synthesis Kit for qPCR uses oligo(dT) to prime cDNA synthesis. Therefore, this kit is best used with intact total RNA preparations.
| ||Figure 1 (click to enlarge). Overview of the MessageBOOSTER™ cDNA Synthesis Kit procedure. The kit uses a linear RNA amplification process to amplify the RNA from a small population of cells, then converts the amplified RNA to cDNA that is ready for PCR. The procedure can be completed in 1 day. |
|Figure 2. Sensitivity of the MessageBOOSTER™ cDNA Synthesis Kit. cDNA produced from 10 pg of total NRK RNA significantly improved the sensitivity (lowered the CT) of detecting the low-abundance PBGD transcript compared to cDNA produced from 10 pg of unamplified RNA. |
|10 pg (~1 cell) ||≥10 ||≥100 |
|100 pg (~10 cells) ||≥100 ||≥1,000 |
|500 pg (~50 cells) ||≥500-1,000 ||≥5,000-10,000 ||Table 1. The number of qPCR amplifications that can be performed using cDNA produced by a MessageBOOSTER™ cDNA Synthesis Kit. The number of qPCRs is dependent on the amount of input total RNA and the abundance of the transcript(s) of interest. |
a. Low-Abundance Transcripts = 1-1,000 copies per cell
Medium-Abundance Transcripts = 1,000-10,000 copies per cell
High-Abundance Transcripts = 10,000-100,000 copies per cell
- Grunenwald, H. et al. (2006) Epicentre Forum 13(3), 22.
- DeFazio, R.A. et al. (2006) J. Neuroscience 26, 3971.
- Kaeffer, B. et al. (2007) Pediatric Research 62, 564.
- Lochner, M., et al. (2008) J. Exp. Med. 205, 1381.
- Graham, D.M. et al. (2008) J. Neurophysiology, 99, 2522.
- Liu, X. et. al. (2009) Am. J. Physiol. Lung Cell Mol Physiol. 296, L158
- Yakovlev, I.A. et al. (2008) Fungal Genetic and Biology 45, 498
- Jung, Y. et al. (2008) Stem Cells Express 10, 1634
- Xi, D. et al. (2008) J. Neurosci Method 10, 1016
- Michel, M-L. et al. (2008) Proc. Nat'l Acad Sci-USA 105(50), 19845
- Satoh-Takayama, N. et al. (2008) Immunity 29, 958
- Bouskra, D. et al. (2008) Nature 10, 1038
- Yakovlev, I.A. et al. (2008) Fungal Genetics and Biology 45, 498
- Roberts, C.D. et. al. (2009) J. Physiology 587, 1657
- Peduto, L. et. al. (2009) J. Immunology 182, 5789
- Xi, D. et. al. (2009) Int'l J. Neuropsychopharmacology, May 13:1-14
MessageBOOSTER™ T7-Oligo(dT) Primer A, RNase Inhibitor, MessageBOOSTER™ Reverse Transcription PreMix, MessageBOOSTER™ DNA Polymerase PreMix 1, MessageBOOSTER™ DNA Polymerase 1, MessageBOOSTER™ cDNA Finishing Solution, MessageBOOSTER™ Random Primers, MessageBOOSTER™ RNase H, MMLV Reverse Transcriptase, MessageBOOSTER™ In Vitro Transcription PreMix A, MessageBOOSTER™ T7 RNA Polymerase, MessageBOOSTER™ T7 Transcription Buffer, RNase-Free DNase I, Forward and Reverse Control PCR Primers, DTT, RNase-Free Water, Poly(I), NRK Total RNA Control.