MonsterScript™ 1st-Strand cDNA Synthesis Kit

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Last modified: 2008-07-15 14:57:15

The MonsterScript™ 1st-Strand cDNA Synthesis Kit is optimized for generating full-length first-strand cDNA from multiple mRNA species in samples containing total cellular or purified polyadenylated RNA.

MonsterScript™ 1st-Strand cDNA Synthesis Kit uses MonsterScript™ Reverse Transcriptase, a reverse transcriptase that lacks RNase H activity. The enzyme's lack of RNase H activity contributes to its ability to make longer cDNAs and more complete libraries of first-strand cDNA molecules. EPICENTRE scientists have demonstrated reverse transcription of 15-kb RNA templates using MonsterScript Reverse Transcriptase.

MonsterScript™ Reverse Transcriptase produces full-length cDNA from mRNA up to or greater than 15 kb The ≈15.2-kb human guanine nucleotide exchange factor, p532, mRNA ('HGNEFp532 mRNA') was reverse transcribed using total HeLa cellular RNA and an oligo(dT)-containing primer in a standard MonsterScript reaction. Two µl of the reverse transcription reaction was used to amplify a 1.3-kb region within 68 bases of the 5'-end of the HGNEFp532 mRNA with the FailSafe™ PCR System. 1A. Detection of the PCR product demonstrates full-length reverse transcription of the HGNEFp532 mRNA by MonsterScript Reverse Transcriptase. 1B. Agarose gel analysis of the PCR reaction shows the 1.3-kb amplicon amplified from the extreme 5'-end of the HGNEFp532 mRNA.

The kit also contains MonsterScript™ RT PreMix, which contains optimized concentrations of dNTPS, Mg⁺² 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 a random nonamer primer.

Figure 2. MonsterScript™ Reverse Transcriptase was used to reverse transcribe six dilutions (8 x 10-6 to 8 x 10-1pg) 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 PCR reactions, in duplicate, using TAQurate™ GREEN Real-Time PCR MasterMix. A PCR efficiency of 99.4% and correlation coefficient of 1.000 was achieved.

Last modified: 2008-05-14 16:46:46

Applications

• First-strand cDNA synthesis
• Production of cDNA 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

Benefits

• MonsterScript™ Reverse Transcriptase lacks RNase H, enabling improved synthesis of full-length cDNA even for long mRNA
• MonsterScript is 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
• Betaine in the cDNA Synthesis PreMix reduces pausing and stops during reverse transcription
• The kit includes both an oligo(dT)-containing and a random nonamer primer
• First-strand cDNA can be made from picogram amounts of total RNA
• A potent RNase Inhibitor is included

Last modified: 2008-05-14 16:46:46

Protocols for: MonsterScript™ 1st-Strand cDNA Synthesis Kit & MonstreScript™ Reverse Transcriptase

(catalogue number MS040910 / MS041050)

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

Last modified: 2008-05-14 16:46:46