One-Step RT-PCR Kit
Designed for simplicity and convenience in carrying out RT-PCR in a one-tube format
The One-Step RT-PCR Kit is designed for simplicity and convenience in carrying out RT-PCR in a one-tube format.
Streamlines and optimizes the RT and PCR steps
Provides a starting point for analysis of new RNA targets
Reverse transcription-polymerase chain reaction, or RT-PCR, is a method for converting and amplifying a single-stranded RNA template to yield abundant double-stranded complementary DNA (cDNA) product (1, 2). In the RT step, the RT enzyme reverse transcribes an RNA template, yielding single-stranded cDNA. In the PCR step, a thermostable DNA polymerase amplifies the single-stranded cDNA to yield double-stranded cDNA product. One step RT-PCR is a variation on the RT-PCR in which all reaction components are mixed in one tube prior to starting the reactions(2). This approach offers simplicity and convenience and minimizes the possibility for contamination.
Complete One-Step RT-PCR Kit
The One-Step RT-PCR Kit comes complete and ready to use for RT-PCR. The kit can be used with diverse RNA samples and custom primers. The kit uses M-MLV Reverse Transcriptase(3) and Taq DNA Polymerase(4), premixed together at concentrations optimized to balance sensitivity and specificity. An optimized reaction buffer, RNase Inhibitor, Ultrapure dNTPs, supplemental magnesium chloride, and RNase-free water are also included, allowing quick, reliable set up of reactions.
Highly Sensitive, Highly Specific
The kit can be used for detection of diverse RNA targets (Fig. 1). Moderately or weakly expressed targets can generally be detected in 1 ng to 1 μg total RNA or 100 pg to 100 ng polyA RNA. Highly expressed targets can be detected in even lower amounts of RNA (Fig. 2). Standard reaction conditions are sufficient for specific amplification of most targets. A few simple protocol adjustments, such as optimization of the amount of primers or addition of supplements for amplifying G+C rich targets, enable specific amplification for others (Fig. 3).
Fig. 1. Amplification of diverse RNA targets by one-step RT-PCR. Target (source): β-actin (100 ng total RNA, human liver), Numb (100 ng total RNA, human liver), Ubiquitin (1 μg total RNA, Arabidopsis leaf), and Terminal Deoxynucleotidyl Transferase (TdT) (100 ng polyA RNA, calf thymus). Gene specific primers were designed to generate products of particular sizes. M: Marker; Lanes 1-3: Actin, 0.5 kb, 1.0 kb, 1.5 kb; Lanes 4-5: Numb, 0.5 kb, 1.0 kb; Lane 6: Ubiquitin, 1.5 kb; Lane 7: TdT, 1.5 kb.
Fig. 2. Highly sensitive detection of β-actin target from human liver total RNA and polyA RNA, by one-step RT-PCR. Primers were used at 0.8μM, a relatively high concentration, in order to achieve high sensitivity. Total RNA: 1 μg to 1 pg, 10-fold dilutions shown in Lanes 1-7. Poly A RNA: 100 ng to 100 fg,
Fig. 3. Flexibility for optimization. For targets with high G+C contents, such as 0.34 kb Notch3 (G+C:77%), adding supplements and/or increasing the temperature of the reverse transcription step, may improve specificity. Compare results for standard reaction conditions versus reaction with betaine supplement and elevated temperature.
Convenient, One-Tube Format
Setting up reactions is quick and simple, given that the RT and PCR steps are set up simultaneously and then carried out sequentially in one tube. This format eliminates the need to set up RT and PCR independently, saving time and eliminating a potential point of contamination.
Rapid, Reliable Results
The One-Step RT-PCR Kit is ideal for qualitative analysis of expression of one or a few genes in multiple RNA samples, analysis of specific RNA splice variants, and streamlining the optimization of RT and PCR steps simultaneously. The kit is also particularly good as a starting point for analysis of new RNA targets, given that many targets can be amplified successfully without need for optimization.
RT-PCR Enzyme Mix
5X RT-PCR Reaction Buffer (includes MgCl2)
Ultrapure PCR Nucleotide Mix (10mM each dATP, dCTP, dGTP, dTTP)
RNase Inhibitor, Recombinant (4 units/μl)
25 mM Magnesium Chloride
- SAMBROOK, J. AND RUSSEL, D. W. (2001) “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 8.46-8.53.
- SELLNER, L. N., COELEN, R. J., AND MACKENZIE, J. S. (1992) Nucleic Acids Res. 20, 1487-1490.
- ROTH, M. J., TANESE, N., AND GOFF, S. P. (1985) J. Biol. Chem. 260, 9326-9335.
- SAIKI, R. K.,GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988) Science 239, 487-491.
- Tech Tip 206, Simple Approaches for Optimization of RT-PCR, USB Corporation, Cleveland, Ohio.