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RNA Amplification for qPCR
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MessageBOOSTERTM

RNA amplification for qPCR

MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit
Applications
  • Amplification of mRNA directly from a cell lysate, without the need for purifying RNA, and conversion of the amplified RNA to cDNA.
  • Significantly increasing the number and sensitivity of qRT-PCRs from very small numbers of cells.
  • Generation of large amounts of cDNA from the mRNA of very small samples for archival purposes.

The MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit enables the user to perform sensitive qRT-PCR studies from very small populations of cells, even from as little as one cell (Table 1).
The kit amplifies the cellular mRNA directly from a cell lysate, without the need to purify total RNA, and then converts the amplified RNA to cDNA that is ready for qPCR (Fig. 1). Amplified cDNA is efficiently produced from the lysates of 1 to 500 cells.

Benefits
  • Obtain up to thousands of qRT-PCRs from as little as one cell.
  • Readily and reproducibly detect even low-abundance transcripts from as little as one cell.
  • No need to purify RNA: Perform RNA amplification and cDNA synthesis reactions directly from cell lysates.
  • High-fidelity, linear RNA amplification process preserves the relative transcript abundance of the sample.
  • Collect samples less often and archive cDNA for future use, saving time and effort.

i_messagebooster_lysates_procedure.gif

Figure 1 (click to enlarge). An overview of the procedure for the MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit. A kit reaction amplifies the poly(A) RNA (mRNA) directly from a crude cell lysate without the need for RNA purification. The amplified RNA is then reverse-transcribed to cDNA that can be diluted up to 1,000-fold for qPCR.

 
c_messagebooster_reaction.gif
  Figure 2. A MessageBOOSTER™ Kit reaction produces sufficient cDNA from a single-cell lysate for thousands of sensitive qPCRs. qPCR was performed using undiluted (red), 1:10 diluted (green), 1:100 diluted (blue), and 1:1,000 diluted (purple) cDNA from a lysate of a single NRK cell. The low-abundance PBGD transcript was readily detected.

MessageBOOSTER Products are covered by intellectual property licensed to Epicentre Technologies Corporation from Johnson & Johnson Pharmaceutical Research & Development, L.L.C., and by intellectual property sublicensed to Epicentre Technologies Corporation from Incyte Corporation.


MessageBOOSTER™ cDNA Synthesis Kit for qPCR

Applications
  • 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.

Benefits
  • Even low-abundance transcripts from total RNA of one cell are readily detected (CT values <35 cycles) (Fig. 2).
  • Collect small RNA samples less often.
  • Reactions can be performed directly from whole-cell lysates without the need for purifying RNA.1
  • Perform a MessageBOOSTER cDNA Synthesis Kit reaction in 1 day.
  • The linear RNA amplification and cDNA synthesis processes preserve the gene expression profile.

i_messagebooster_cdnasynthesiskit.gif

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.

c_messagebooster_cdnasensitivity.gif
  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.
 스크린샷 2013-06-13 오후 2.07.56.png  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
Product Citations
  1. Grunenwald, H. et al. (2006) Epicentre Forum 13(3), 22.
  2. DeFazio, R.A. et al. (2006) J. Neuroscience 26, 3971.
  3. Kaeffer, B. et al. (2007) Pediatric Research 62, 564.
  4. Lochner, M., et al. (2008) J. Exp. Med. 205, 1381.
  5. Graham, D.M. et al. (2008) J. Neurophysiology, 99, 2522.
  6. Liu, X. et. al. (2009) Am. J. Physiol. Lung Cell Mol Physiol. 296, L158
  7. Yakovlev, I.A. et al. (2008) Fungal Genetic and Biology 45, 498
  8. Jung, Y. et al. (2008) Stem Cells Express 10, 1634
  9. Xi, D. et al. (2008) J. Neurosci Method 10, 1016
  10. Michel, M-L. et al. (2008) Proc. Nat'l Acad Sci-USA 105(50), 19845
  11. Satoh-Takayama, N. et al. (2008) Immunity 29, 958
  12. Bouskra, D. et al. (2008) Nature 10, 1038
  13. Yakovlev, I.A. et al. (2008) Fungal Genetics and Biology 45, 498
  14. Roberts, C.D. et. al. (2009) J. Physiology 587, 1657
  15. Peduto, L. et. al. (2009) J. Immunology 182, 5789
  16. Xi, D. et. al. (2009) Int'l J. Neuropsychopharmacology, May 13:1-14
*MessageBOOSTER Products are covered by intellectual property licensed to Epicentre Technologies Corporation from Johnson & Johnson Pharmaceutical Research & Development, L.L.C., and by intellectual property sublicensed to Epicentre Technologies Corporation from Incyte Corporation.


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