Gene Expression Analysis |
Reverse Transcription and cDNA Synthesis
Order
| Protocol
| Citations
Applications
- First-strand cDNA synthesis for subsequent PCR or real-time PCR.
- RT-PCR validation of gene expression data obtained from microarray experiments.
- RT-PCR validation and quantification of gene silencing by RNA interference.
- Preparation of labeled cDNA probes.
MonsterScript™ Reverse Transcriptase is a thermostable reverse transcriptase
that completely lacks RNase H activity. The enzyme's lack of RNase H activity
contributes to its ability to make longer cDNAs and more complete full-length
libraries of first-strand cDNA molecules compared to other reverse transcriptases.
EPICENTRE scientists have demonstrated reverse transcription of 15-kb RNA templates
using MonsterScript™ Reverse Transcriptase (Fig. 1).
The MonsterScript 1st-Strand cDNA Synthesis Kit includes MonsterScript RT
PreMix, which contains optimized concentrations of dNTPs, Mg2+, and
betaine.* Betaine reduces pausing and stops by the reverse transcriptase, enabling
improved reverse transcription through difficult sequences, such as regions
of high GC content. The kit also contains both an oligo(dT)-containing primer
and random nonamer primers.
Benefits
- No RNase H activity, enabling improved synthesis of full-length cDNA even
for long mRNA, using random priming.
- Thermostable, permitting reverse transcription at temperatures >50°C,
which reduces RNA secondary structure and improves priming specificity.
- MonsterScript RT PreMix contains optimized concentrations of dNTPs, Mg2+,
and betaine for superior performance and minimal pipetting steps.
- The kit includes both an oligo(dT) and random nonamer primers for maximal
transcript coverage.
- First-strand cDNA can be made from picogram amounts of total RNA (Fig. 2).
- The kit includes a potent RNase Inhibitor to protect the integrity of template
RNA.
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| Figure 1. MonsterScript™ Reverse Transcriptase
produces full-length cDNA from mRNA greater than 15 kb. The ≈15.2-kb
HGNEFp532 mRNA was reverse-transcribed from total HeLa RNA in a standard
MonsterScript reaction. Two microliters of the reaction was used to PCR amplify
a 1.3-kb region within 68 bases of the 5´ end of the HGNEFp532 mRNA
(A). Agarose gel electrophoresis of the 1.3-kb amplicon from the 5´ end
of the mRNA demonstrates full-length cDNA synthesis (B). |
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Figure 2. MonsterScript™ Reverse Transcriptase
was used to reverse-transcribe six dilutions (8 x 10-6 to 8 x
10-1 pg) of an RNA transcript into cDNA in 20-µl reactions. Ten
microliters of each reverse transcription reaction was then used in 50-µl
real-time PCRs, in duplicate, using EPICENTRE's TAQurate™ GREEN Real-Time
PCR MasterMix. A PCR efficiency of 99.4% and correlation coefficient of 1.000
was achieved. |
Product Citations
- Alice, A.F. et al. (2006) J. Bacteriol. 188, 1551.
- Budde, P.P. et al. (2007) J. Bacteriol. 189, 491.
- Conejo, M. et al. (2008) J. Molec. Evolution 66, 11.
- Chumely, M.J. et al. (2007) J. Neurosci. 27, 13481.
- Borchert, G.M. (2006) Nature Struct. Mol. Biol. 13, 1097.
*Covered by issued and/or pending patents.
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