Cambio - Excellence in Molecular Biology

Gene Expression Analysis

Gene Expression Analysis: Gene Expression Analysis

MessageBooster™ cDNA Synthesis Kit for qPCR

The MessageBOOSTER™ cDNA Synthesis Kit for qPCR greatly improves qRT-PCR sensitivity, accuracy, and reproducibility of even low-abundance transcripts from as little as 1 cell

BioSearch Tech (Lucigen/Epicentre)

Description

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.
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.

 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.
Amount of Total RNA in a MessageBOOSTER™ Reaction Number of qPCRs That Can Be Performed
Low- to Medium- Abundance Transcriptsa Medium- to High- Abundance Transcriptsa
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

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

 

 




If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

Protocols

Protocols for: MessageBOOSTER™ cDNA Synthesis Kit for qPCR 

Due to the constant updating of the protocols by the manufacturer we have provided a direct link to Lucigen’s product page, where the latest protocol is available.

Please note this will open a new page or window on your computer.

 MessageBOOSTER™ Protocol

(catalogue number  MB060124) 

Please note: all protocols off site are the responsibility of the products supplier

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

References

References

    1. DeFazio RA, et al. (2006) J. Neuroscience 26(15) 3971
    2. Kaeffer, B. et. al. (2007) Pediatric Research 62 (5): 564.
    3. Graham, D.M., et. al. (2008)J. Neurophysiol. v.99
    4. Lochner, M., et. al. (2008) J. Exp. Med., 10.1084/jem.20080034

 

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

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

Applications & Benefits

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.

Benefits

  • Even low-abundance transcripts from total RNA of one cell are readily detected (CT values <35 cycles).
  • 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.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

Related products

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200