FAQs

Access RT-PCR and AccessQuick™ Systems

See Technical Resources for more information.

1. What is RT-PCR?
2. What are the Access and AccessQuick™ RT-PCR Systems?
3. Can total RNA be used as template, or is poly(A)+ RNA required?
4. How much RNA is required for the Access or AccessQuick™ RT-PCR Systems?
5. What advantages do the Access and AccessQuick™ RT-PCR Systems have compared to other commonly used methods?
6. What conditions may require optimization when using the Access or AccessQuick™ RT-PCR System?
7. Does Tfl DNA Polymerase have any reverse transcriptase activity?
8. Can MgCl₂ be substituted for the MgSO₄ provided with the Access RT-PCR System?

1. What is RT-PCR?
   RT-PCR is a technique that couples reverse transcription (RT) of an RNA template with polymerase chain reaction (PCR) amplification of the resulting cDNA. RT-PCR is a sensitive and versatile method that can be used to detect the presence of a transcript, estimate expression levels and clone cDNA products without constructing and screening a cDNA library. For more detailed information, see the Promega Protocols and Applications Guide and RNA Analysis Notebook.

2. What are the Access and AccessQuick™ RT-PCR Systems?
   The Access RT-PCR System (Cat.# A1250) and AccessQuick™ RT-PCR System (Cat.# A1701) allow reverse transcription and PCR amplification of a specific target RNA from total RNA or mRNA. The one-tube, two-enzyme systems provide sensitive, quick and reproducible analysis of even rare RNAs. The systems use Avian Myeloblastosis Virus (AMV) Reverse Transcriptase for first-strand cDNA synthesis and the thermostable Tfl DNA Polymerase from Thermus flavus for second-strand cDNA synthesis and DNA amplification.
   
   The Access RT-PCR System includes the RT-PCR reagents as separate components, so you have maximum control when optimizing amplification conditions, such as Mg²⁺ concentration. The AccessQuick™ RT-PCR System has an easy and convenient master mix, which includes Tfl DNA Polymerase, dNTPs, magnesium chloride and reaction buffer, to increase the convenience of performing RT-PCR. Importantly, the AMV RT enzyme is provided in a separate tube to allow no-RT control reactions.

3. Can total RNA be used as template, or is poly(A)+ RNA required?
   The RNA template may be total RNA, mRNA [poly(A)+] or a synthetic RNA transcript. Successful reverse transcription depends on RNA template integrity and purity. Regardless of the type of RNA used, it is critical that an RNase-free environment be maintained during RNA isolation. The PureYield™ RNA Midiprep System, (Cat.# Z3740), SV Total RNA Isolation System (Cat.# Z3100), RNAgents^® Total RNA Isolation System (Cat.# Z5110), MagneSil^® Total RNA mini-Isolation System (Cat.# Z3351) and PolyATtract^® mRNA Isolation System (Cat.# Z5200) yield RNA of sufficient purity for use in the Access and AccessQuick™ RT-PCR Systems. Additionally, care should be taken to minimize the amount of DNA present in the RNA preparation. Treatment of RNA samples with RQ1 RNase-Free DNase (Cat.# M6101), followed by extraction and precipitation, is recommended for RNA samples that might contain DNA.

4. How much RNA is required for the Access or AccessQuick™ RT-PCR Systems?
   The minimum amount of RNA that can be amplified using these systems is both template- and primer-dependent. With both systems, excellent amplification results can be obtained using total RNA template levels in the range of 10pg–1µg per reaction or poly(A)+ RNA template levels in the range of 1pg–100ng.

5. What advantages do the Access and AccessQuick™ RT-PCR Systems have compared to other commonly used methods?
   For both systems, reactions are performed in a single tube without secondary additions to the reaction mix. This decreases hands-on time relative to some commonly used methods and minimizes the likelihood of introducing contaminants into the reaction. Furthermore, these two-enzyme systems are more sensitive than a single-enzyme rTth DNA polymerase-based system for amplification of a target in a total RNA preparation. In addition, the Access RT-PCR System includes a Positive Control RNA, which can help ensure reactions were assembled correctly and troubleshoot failed amplifications. Unlike many commercially available RT-PCR systems that include a master mix, the AccessQuick™ RT-PCR System has a separate tube of AMV RT so that no-RT control reactions can be assembled; performing a no-RT control reaction is important because absence of the desired band in the no-RT control reaction confirms that the product was amplified from an RNA template, not a DNA template.

6. What conditions may require optimization when using the Access or AccessQuick™ RT-PCR System?
   There are several parameters that can be optimized for a particular primer-target combination, including MgSO₄ concentration, primer annealing temperature and number of amplification cycles.

   • The magnesium requirement of both the AMV Reverse Transcriptase and Tfl DNA Polymerase in the Access and AccessQuick™ RT-PCR System reactions is affected by the final concentration of nucleotides, oligonucleotide primers and template. For the Access RT-PCR System, we recommend testing MgSO₄ concentrations from 0.5–3.0mM (in 0.5mM increments) initially. For the AccessQuick™ RT-PCR System, the final concentration of MgSO₄ concentration in the reaction is 1.5mM; we recommend testing MgSO₄ concentrations from 1.5–3.0mM in 0.5mM increments.
   • For primers with a high melting temperature increasing the suggested annealing and extension temperatures may be advantageous. The higher temperature minimizes nonspecific primer annealing, thus increasing the amount of specific product.
   • Most RNA targets can be detected after 40 cycles of PCR amplification. However, if the target is rare or if only a small amount of starting material is available, it may be necessary to increase the number of cycles to 45 or 50.

   More information about optimizing RT-PCR amplifications can be found in the Promega Protocols and Applications Guide.

7. Does Tfl DNA Polymerase have any reverse transcriptase activity?
   Tfl DNA Polymerase exhibits no endogenous reverse transcriptase activity under the assay conditions tested.

8. Can MgCl₂ be substituted for the MgSO₄ provided with the Access RT-PCR System?
   MgCl₂ should not be substituted for the MgSO₄ provided. Tfl DNA Polymerase exhibits greatly enhanced performance using MgSO₄ and the AMV/Tfl 5X Reaction Buffer under the suggested assay conditions.