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MonsterScriptTM

cDNA Synthesis Kit

MonsterScript™ 1st-Strand cDNA Synthesis Kit

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, Mg
  • 2+, 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.

g_monsterscript_fulllengthcdna.jpg
 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).

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.
c_monsterscript_revtranscribe.gif 

Product Citations
  1. Alice, A.F. et al. (2006) J. Bacteriol. 188, 1551.
  2. Budde, P.P. et al. (2007) J. Bacteriol. 189, 491.
  3. Conejo, M. et al. (2008) J. Molec. Evolution 66, 11.
  4. Chumely, M.J. et al. (2007) J. Neurosci. 27, 13481.
  5. Borchert, G.M. (2006) Nature Struct. Mol. Biol. 13, 1097.
*Covered by issued and/or pending patents.


MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit 

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.
The MMLV High Performance Reverse Transcriptase (MMLV HP RT) and the MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit both produce full-length first-strand cDNA from total cellular RNA preparations or purified poly(A) RNA.
MMLV HP RT demonstrates significantly greater reverse transcriptase activity than other commercially available MMLV RT enzymes. Typically, just 100 units of MMLV HP RT are required for full-length cDNA synthesis compared to 200 units of competitive MMLV RT enzymes. MMLV HP RT includes a 10X Reaction Buffer, optimized for synthesis of full-length cDNA from long RNA templates, and DTT.

g_mmlv_full-length_cdna_from_rna.gif
Figure 1. MMLV HP RT produces full-length cDNA from mRNA longer than 15 kb. Total RNA isolated from HeLa cells was reverse transcribed and the cDNA was amplified by PCR. Detection of the 1.3-kb PCR amplicon from near the 5´ end of the mRNA demonstrates full-length reverse transcription of HERC1 mRNA (A). Agarose gel analysis of the PCR products shows the 1.3-kb amplicon from the 5´ end of the mRNA (B). Lane M, 100 bp DNA ladder; lane 1, no-RT control reaction; lane 2, PCR product from cDNA synthesized by Epicentre's MMLV HP RT.

Benefits
  • Synthesize full-length cDNA from RNA templates longer than 15 kb.
  • MMLV HP RT demonstrates significantly higher activity than competitive reverse transcriptase enzymes.
  • Reaction Buffer, optimized for producing full-length cDNA, is included with both the MMLV HP RT and the MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit.
  • The kit includes both an oligo(dT) and random nonamer primers.
  • First-strand cDNA can be made from picogram amounts of input total RNA.
  • The kit includes a potent RNase Inhibitor to protect the integrity of template RNA.

스크린샷 2013-06-13 오후 5.19.06.png
Table 1. 3´/5´ ratio analysis of cDNA produced by different reverse transcriptase enzymes. Total cellular RNA from HeLa cells was converted to cDNA using the three reverse transcriptase enzymes indicated in the table. A 3´/5´ ratio equal to 1.0 means that equal amounts of PCR products are obtained from both the 3´ and 5´ end of the cDNA and therefore is a good indication that the reverse transcriptase has produced a full-length cDNA copy of the mRNA.
 2A.
mmlv_i2140703.gif
 Figure 2. cDNA produced by Epicentre's MMLV High Performance Reverse Transcriptase yields a significantly improved 3´/5´ ratio than competitive reverse transcriptases. The approximately 2160-base HeLa β-glucuronidase mRNA (GUSB) was reverse transcribed into cDNA using Epicentre's MMLV High Perfomance Reverse Transcriptase and two competitive reverse transcriptase enzymes. PCR primer pairs to the 3´-end and 5´-end of GUSB cDNA were synthesized and qPCR (SYBR® Green I dye detection) was performed using each primer pair and the GUSB cDNAs as templates. The 3´/5´ ratio was calculated for each as described in the text. (A). PCR amplicons from the 3´ end and 5´ end of the GUSB cDNA. (B). qPCR quantification graphs for detecting the 3´ amplicon and 5´ amplicon of GUSB cDNA produced by Epicentre's MMLV High Performance Reverse Transcriptase Kit and two other commercially available reverse transcriptase enzymes.

2B
 Epicentre MMLV 1st-Strand cDNA Synthesis Kit
 Company I RNase H- minus MMLV RT
 
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 Company P MMLV RT
 
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RNase-Free DNase I

Applications
  • Elimination of template DNA following in vitro synthesis of RNA with T7, SP6, or T3 RNA polymerase.
  • Labeling of DNA by nick translation, in combination with Klenow or other DNA polymerases.
  • Treatment of RNA prior to RT-PCR.1
  • Characterization of DNA-protein interactions by DNase I footprinting.2,3
RNase-Free DNase I (bovine pancreas) is an endonuclease useful in removing DNA that might interfere with the characterization, manipulation, or use of RNA, or for any application requiring highly purified DNase I, such as nick translation. It efficiently hydrolyzes dsDNA and ssDNA into a mixture of short oligonucleotides and mononucleotides.

dnase_1.gif
Figure 1. DNA removal from in vitro transcription reactions using RNase-Free DNase I. A linearized DNA template was transcribed using T7 RNA polymerase according to standardin vitro transcription conditions. Lane 1, kb ladder; Lane 2, DNA control; Lane 3, transcription mixture; Lane 4, transcription mixture treated with 1 MBU of RNase-Free DNase I for 15 minutes at 37°C.

Unit Definition: One Molecular Biology Unit (MBU) of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutes at 37°C under standard assay conditions.

Storage Buffer: 50% glycerol containing 10 mM Tris-HCl (pH 7.5), 10 mM CaCl2, and 10 mM MgCl2.

DNase I 10X Reaction Buffer: 100 mM Tris-HCl (pH 7.5), 25 mM MgCl2, and 5 mM CaCl2.

Quality Control: No degradation of 1 µg of a synthetic RNA transcript is detected by agarose gel electrophoresis following incubation with 10 U of RNase-Free DNase I at 37°C for 1 hour.

References
  1. Kienzle, N. et al. (1996) BioTechniques 20, 612.
  2. Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.) Cold Spring Harbor Laboratory Press, New York.
  3. Galas, D.J. and Schmitz, A. (1978) Nucleic Acids Res. 5, 3157
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