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Performance Comparisons of Commercial RT-PCR Systems


prostar RT-PCR systems excel at amplifying challenging targets

Michael Borns Janice Cline Holly Hogrefe
Stratagene

We compared Stratagenes prostar RT-PCR systems to other commercially available RNA amplification kits to see how well they performed. The ProSTAR HF single-tube RT-PCR system, featuring the TaqPlus Precision DNA polymerase blend * and a convenient one-step, one-tube format, provides greater target-length capability and higher product yields than other one-tube RT-PCR kits. The ProSTAR ultra HF RT-PCR system, featuring PfuTurbo DNA polymerase** and a versatile two-step format, amplifies the longest targets and produces the lowest mutation frequencies of any RNA amplification system tested. The superior accuracy of the ProSTAR ultra HF RT-PCR system is directly related to the use of PfuTurbo DNA polymerase for cDNA amplification.

Stratagene recently introduced the ProSTAR line of RNA amplification reagents, featuring the ProSTAR HF (high fidelity) single-tube and the ProSTAR ultra HF (ultra-high fidelity) RT-PCR systems.1 The ProSTAR HF single-tube RT-PCR system combines the sensitivity and accuracy of the TaqPlus Precision DNA polymerase blend2 with the convenience of a one-step, one-tube procedure. With the ProSTAR ultra HF RT-PCR system, a versatile two-step format is used, which features PfuTurbo DNA polymerase,3 a special formulation of Pfu DNA polymerase and a novel PCR-enhancing factor*** for amplifying cDNAs with the highest replication fidelity possible. In a previous Strategies article, it was demonstrated that the ProSTAR RT-PCR systems are useful in applications requiring robust, sensitive, high-fidelity amplification of RNA from a variety of RNA sources.1

Here, we compare the performance of Stratagenes ProSTAR RT- PCR systems to other commercial RT-PCR kits with respect to product yield, maximum length of products amplified, ease of use, and accuracy.

Comparing One-Tube RT-PCR Kits

Figure 1 shows results from amplifying a series of primer-template systems using the ProSTAR HF single-tube RT-PCR system and one-tube RNA amplification systems/kits from other vendors. RT-PCR reactions were carried out under similar conditions, according to each manufacturers recommendations. With all kits, we successfully amplified the shortest 1.8-kb target (Panel A). Only the ProSTAR HF single-tube RT-PCR system and the Titan One-Tube RT-PCR kit consistently amplified the longer 3.6-kb (Panel B) and 4.1-kb (Panel C) targets. Product yields, however, were consistently higher with the ProSTAR HF single-tube RT-PCR system (Figure 1, Panels A, B, and C; Lanes 1). Observed differences in the migration of RT-PCR products produced with the ProSTAR HF single-tube and Titan One-Tube RT-PCR systems are most likely due to variation in buffer composition.

In addition to superior product yields, the ProSTAR HF single-tube RT-PCR system is more convenient and versatile, in contrast to other suppliers kits. For example, the performance of the Titan One-Tube RT-PCR system was dependent upon the use of a specific cycling regimen (data not shown), which requires progressive increases in extension time per cycle (Methods). Therefore, realizing optimal performance of the Titan kit is limited to customers with temperature cyclers that can be programmed in this fashion. Maximum performance is achieved with the ProSTAR HF single-tube RT-PCR system using both fixed and progressively lengthened extension times (data not shown).

The LA RNA PCR kit amplified two of the three targets tested. It should b e pointed out, however, that this kit is not truly an optimized one-step RT-PCR kit, like the ProSTAR HF single-tube RT-PCR system. The LA RNA PCR kit permits cDNA synthesis and PCR amplification in the same tube, but secondary reagents must be added between the RT and PCR amplification steps or a wax barrier must be used to exclude PCR reagents from the cDNA synthesis reaction. In contrast, the ProSTAR HF RT-PCR system provides for cDNA synthesis and PCR amplification in a common RT-PCR buffer during an uninterrupted thermal cycling program. This makes the ProSTAR HF RT-PCR system easier to use and less prone to template contamination, while providing consistently higher product yields. Finally, the Access RT-PCR system features a versatile one-step format like the ProSTAR HF single-tube RT-PCR system. However, because the target-length capability of the Access RT-PCR system is limited, its utility is restricted in demanding RNA amplification applications. Additionally, in the Access RT-PCR system, cDNA amplification is carried out with Tfl DNA polymerase, which may limit the kits performance compared to other kits that employ more robust DNA polymerase blends (e.g., ProSTAR HF, Titan, and LA RNA PCR systems/kits).

Comparing Two-Tube RT-PCR Kits

Figure 2 shows results from amplifying a series of primer-template systems using the ProSTAR ultra HF RT-PCR system and two-tube RNA amplification systems/kits from other vendors. RT-PCR reactions were carried out under similar conditions, according to each manufacturers recommendations. The ProSTAR ultra HF RT-PCR system consistently amplified all three targets in high yield, including a relatively long amplicon that was 7.6 kb in length (Panel C). The ThermoScript RT-PCR system with Platinum Taq DNA Polymerase High Fidelity also amplified all three targets. However, product yields were consistently higher with the ProSTAR ultra HF RT-PCR system under more demanding conditions, where longer, low-abundance targets are amplified (Figure 2, Panel C). Using Advantage RT-for-PCR kit reagents, successful amplification was not achieved for any of the targets we tested.

prostar Ultra HF RT-PCR System Provides the Most Accurate RNA Amplification

The accuracy of the ProSTAR ultra HF RT-PCR system was compared to other commercial RNA amplification kits. A PCR-based forward mutation assay4 was modified to allow amplification of the lacI target gene from cDNA synthesized from in vitro-transcribed mRNA. The lacI-containing RT-PCR products were produced with various RNA amplification systems/kits using conditions that were as similar as possible, while following the guidelines specified by each manufacturer (Figure 3 legend). RT-PCR products were cloned into lambda arms, and the percentage of lacI mutants was determined in a color-screening assay. Separate controls, in which the RT was omitted, were performed for each kit. Results indicated that the RT-PCR products used in the fidelity assay were synthesized from reverse-transcribed cDNA, not from contaminating genomic DNA template.

Fig.3

Of the RT-PCR systems/kits tested, mutation frequencies were found to be lowest in RT-PCR reactions carried out with the ProSTAR ultra HF RT-PCR system (Figure 3). The percentage of mutant clones was significantly greater for all other RNA amplification systems/kits, irrespective of the reverse transcriptase, cDNA synthesis temperature, or kit format employed. The greater accuracy of the ProSTAR ultra HF RT-PCR system is related to the use PfuTurbo DNA polymerase for cDNA amplification. Additional results showed that cDNA produced with different RTs (37-65C) was amplified more accurately with PfuTurbo DNA polymerase than with Tfl, Taq, or Taq-based DNA polymerase blends (Platinum Taq High Fidelity, Advantage KlenTaq Polymerase mix, Expand High Fidelity, LA DNA polymerase blend) (Figure 3 and data not shown). Therefore, PfuTurbo DNA polymerase is the superior choice for carrying out RNA amplification procedures that require the greatest accuracy possible, such as cDNA cloning, sequencing, and/or expression.

Conclusions

Stratagene's ProSTAR RT-PCR systems provide exceptional performance, compared to other commercially available RNA amplification kits. The ProSTAR HF single-tube RT-PCR kit offers the convenience and speed of a one-step format, while consistently producing higher product yields and longer target amplification. The ProSTAR ultra HF RT-PCR system provides superior yield and target-length capability in a versatile two-step format, and the inclusion of PfuTurbo DNA polymerase allows RNA amplification with the greatest accuracy possible.

Methods

RT-PCR reactions were carried out using the recommended protocols for each RNA amplification system/kit. For the ProSTAR HF single-tube RT-PCR system, 50-l RT-PCR reaction mixtures contained 1X HF RT-PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l each), RNA (200 ng of total RNA), 1.25 U of MMLV-RT, and 2.5 U of TaqPlus Precision DNA polymerase. Amplifications were carried out in Stratagenes RoboCycler gradient 96 temperature cycler fitted with a Hot Top assembly, using 200-l thin-walled PCR tubes. The temperature cycling parameters for all tar gets incorporated the following. For cDNA synthesis, cycling included one cycle at 37C for 15 minutes (1.8-kb target) or 30 minutes (3.6- and 4.1-kb targets). For PCR, cycling included one cycle at 95C for 1 minute, followed by 40 cycles at 95C for 1 minute (denaturation); 60C for 1 minute (annealing); 68C for 2 minutes (1.8-kb target) or 2 minutes/kb (3.6, 4.1-kb) (extension); and one final extension cycle of 68C for 10 minutes.

For the ProSTAR ultra HF RT-PCR system, 10-l cDNA synthesis reactions contained 1X MMLV-RT buffer, 1 mM each dNTP, 10 ng/l oligo(dT), and RNA (200 ng of total RNA). The reaction mixtures were incubated at 65C for 5 minutes and cooled to room temperature to allow the primers to anneal to the RNA. MMLV-RT (10 U) was added, and cDNA synthesis was carried out at 37C for 15 minutes (0.3- and 1.8-kb targets) or 30 minutes (7.6-kb target). PCR amplification reactions (50-l) contained 1X ultra-HF PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l each), 1 l of cDNA, and 2.5 U of PfuTurbo DNA polymerase. PCR amplification was performed as described above, except that the extension at 68C was carried out for 3 minutes/kb.

Performance comparisons were carried out between the ProSTAR HF single-tube RT-PCR system and the following one-step, one-tube RT-PCR kits: Boehringer Mannheims Titan One-Tube RT-PCR system (AMV RT, Expand High Fidelity enzyme blend), Takaras LA RNA PCR kit (AMV RT, LA DNA polymerase blend), and Promegas Access RT-PCR system (AMV RT, Tfl DNA polymerase). RT-PCR reactions (50-l reactions) were assembled according to each kits recommended procedure, using the same primer and template amounts (see above). The cDNA synthesis and temperature cycling conditions used were those recommended by the kit manufacturer: With the Titan RT-PCR system, cDNA synthesis was carried out as follows: 50C for 30 minutes and (denaturation) 94C for 2 minutes. For PCR, incubations were 94C for 30 seconds, 60C for 30 seconds, and 68C for 1 minute/cycle (1.8-kb target) or 2.5 minutes/cycle (3.6-kb target) or 3 minutes/cycle (4.1-kb target) (10 cycles); 94C for 30 seconds, 60C for 30 seconds, and 68C for 1 minute plus 5 seconds/cycle (1.8-kb target) or 2.5 minutes plus 5 seconds/cycle (3.6-kb target) or 3 minutes plus 5 seconds/cycle (4.1-kb target) (25 cycles; an additional 5 seconds was added progressively to the 68C extension step at each cycle); the final extension was 7 minutes (one cycle). With the LA RNA PCR kit, a one-step RT-PCR was carried out using the AmpliWax PCR Gem 100 (Applied Biosystems) protocol and the following conditions for cDNA synthesis: 30C for 10 minutes, 55C for 30 minutes, and (denaturation) 95C for 5 minutes. For PCR, incubations were 94C for 30 seconds and 65C for 5 minutes (30 cycles). With Promegas Access RT-PCR system, cDNA synthesis included incubations at 48C for 45 minutes and (denaturation) 94C for 2 minutes; for PCR, incubations were 94C for 30 seconds, 60C for 1 minute, and 68C for 2 minutes/kb (40 cycles).

Performance comparisons were carried out between the ProSTAR ultra HF RT-PCR system and the following two-tube RT-PCR kits: Life Technologies, Incorporateds ThermoScript RT-PCR system (ThermoScript RT, Platinum Taq DNA Polymerase High Fidelity) and Clontechs Advantage RT-for-PCR kit (MMLV RT, Advantage KlenTaq Polymerase Mix). All cDNA synthesis (20 l) and PCR amplification (50 l) reactions were assembled according to each kits recommended procedure, using the same primer and template amounts (see above). The cDNA synthesis and temperature cycling conditions used were those recommended by the kit manufacturer. With the ThermoScript RT-PCR system, cDNA synthesis was as follows: 55C for 30 minutes (0.3- and 1.8-kb targets) or 1 hour (7.6-kb target), then (denaturation) 94C for 2 minutes. For PCR, incubations were 94C for 30 seconds, 60C for 1 minute, and 68C for 2 minutes/kb (40 cycles). With Advantage RT-for-PCR kit, cDNA synthesis was as follows: 42C for 1 hour and (denaturation) 94C for 1 minute. For PCR, incubations were 94C for 45 seconds, 60C for 1 minute, and 68C for 2 minutes/kb (40 cycles).

RT-PCR products were electrophoresed on 0.8 to 1% agarose/1X TBE gels, stained with ethidium bromide, and imaged using the Eagle Eye II still video system.

REFERENCES

  1. Borns, M., et al. (1999) Strategies 12: 33-36.

  2. Nielson, K.B., et al. (1997) Strategies 10: 29-32.

  3. Hogrefe, H., et al. (1997) Strategies 10: 93-96.

  4. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucl. Acids Res. 24: 3546-3551.

* U.S. Patent No. 5,556,772 and patents pending
** U.S. Patent Nos. 5,545,552 and 5,866,395 and patents pending
*** Patents pending


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