PyroScript® RT-PCR 2X Master Mix Kit

The PyroScript® RT-PCR 2X Master Mix Kit incorporates a thermostable PCR enzyme discovered by Lucigen that also has efficient reverse transcription activity. PyroScript RT-PCR 2X Master Mix simplifies RNA detection by allowing accurate single-tube, single-enzyme reverse transcription PCR (RT-PCR) of amplicons up to 400 bp. This format reduces hands-on time and facilitates error-free reaction set up. Just add template, primers and cycle. Detect amplification by gel electrophoresis or real-time analysis.

Low background fluorescence and enzyme compatibility with commonly used fluorescent stains make PyroScript RT-PCR 2X Master Mix excellent for qRT-PCR. PyroScript RT-PCR 2X Master Mix is especially useful for RT-PCR detection and quantification of viral as well as transcript RNA. Extreme thermostability allows denaturation of crude specimens at 94°C to simplify sample preparation. Reverse transcription at 70°C or even higher improves analysis of highly-structured RNA templates.

Amplify up to 400 bp of transcript RNA and viral RNA

PyroScript RT-PCR 2X Master Mix was used to detect Human 18S rRNA sequences and  viral RNA, as shown in figure 1.

Figure 1. Single Enzyme RT-PCR A.) Human 18S rRNA sequences were amplified from 100 pg total A549 cell line RNA. Five primer sets targeting amplicons of between 51 and 65% GC content and from 87 to 367 bp in length were tested. B.) Viral RNA (Enterobacteriophage MS2, ATCC 15597-B1) was amplified by 40 cycles of RT-PCR without background.  Products from 89 to 362 bp in length were amplified. One-step single-enzyme RT-PCR cycle conditions: 15 sec @ 94°C (10s @ 94°C, 30s @ 72°C)*40.

PyroScript™ RT-PCR 2X Master Mix is Sensitive

The sensitivity of PyroScript RT-PCR 2x Master Mix is shown in figure 2, in which as few as 1.2 copies of MS2 bacteriophage RNA were detected.

Figure 2. Single-enzyme, one-step RT-PCR from a 10-fold dilution series of quantitated MS2 bacteriophage RNA. Pyroscript RT-PCR Master Mix amplified RNA copy numbers from 120,000 to 1.2 copies (as estimated by OD260). No amplification was detected in the water only no target control (NTC).

Linear RT-PCR Quantification with PyroScript RT-PCR 2X Master Mix

PyroScript RT-PCR 2x Master Mix was used in real time analysis, as shown in figure 3, to illustrate the linearity of quantification.

Figure 3. Quantitative RT-PCR Triplicate single-enzyme RT-PCR amplifications of a 103 to 108-fold dilution series of the RNA target. Amplification was detected by EvaGreen fluorescence. PyroScript RT-PCR 2X Master Mix, RNA Control and Control Primer Set were used for the analysis.

PyroScript RT-PCR 2X Master Mix outperforms Tth for single enzyme RT-PCR

As shown in figure 4, PyroScript RT-PCR 2X Master Mix detects an RNA target with much greater sensitivity and far less primer dimer formation than RT-PCR using Tth polymerase.

Figure 4. Single-enzyme, one-step RT-PCR of a 160 bp amplicon using a 102 to 108-fold dilution series of MS2 RNA. Tth polymerase used with Mn2+ as directed (Epicentre). Arrow shows amplicon.

Legal Information

The methods of using the PyroScript RT-PCR 2X Master Mix Kit are covered by pending patents assigned to Lucigen Corporation. Lucigen does not encourage or support the unauthorized or unlicensed use of the PCR process, reverse transcription PCR process or isothermal DNA synthesis. It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties.

PyroScript RT-PCR 2X Master Mix and Quality Control Assays

PyroScript RT-PCR 2X Master Mix: Polymerase enzyme and 2X buffer including stabilizers, 40 mM monovalent salts, 0.2 mM each dNTP (N=A,C,G,T), and 4 mM MgSO₄.

Activity Assay: Polymerase activity is assayed at 70°C with 0.2 mM each of dATP, dGTP, dTTP, dCTP (mix of unlabeled and [33P] dCTP); 10 µg activated calf thymus DNA, and 0.1 mg/ml BSA in a final volume of 50 µl.

Absence of Endonuclease: Polymerase is determined to be free of detectable endonuclease or nicking activity. One µg of supercoiled plasmid DNA is incubated with enzyme for 16 hours at 70°C. Agarose gel electrophoresis shows no alteration in mobility consistent with endonuclease or nicking activity.

Absence of Exonuclease: Polymerase is tested to be free of contaminating exonuclease activity by incubating 1 µg of Hind III-digested lambda DNA with enzyme at 70°C for 16 hours. Agarose gel electrophoresis shows no alteration in mobility consistent with exonuclease activity.

Absence of Ribonuclease: Polymerase and PyroScript RT-PCR 2X Master Mix are tested to be free of contaminating RNAse activity by incubating with a fluorogenic RNAse substrate for 1 hour at 37°C. No increase in assay fluorescence above background is detected.

Functional Assays: PyroScript RT-PCR 2X Master Mix is tested for performance in PCR by amplification of a 2 kb region of the endA gene from 2 × 10⁶ molecules (10 ng) of E. coli gDNA. The resulting PCR product is visualized on an ethidium bromide-stained agarose gel.

PyroScript RT-PCR 2X Master Mix is tested for performance in RT-PCR with Control Primer Set I to amplify a 160 bp region of the RNA Control I. The RT-PCR is tested for linearity by RT-qPCR and amplification products are visualized on an ethidium bromide-stained 2% agarose gel.