The Two-Step RT-PCR Kit is designed for flexibility and versatility in carrying out RT-PCR in one or two-tube formats.
- Qualitative analysis of gene expression
- Highly detailed analysis of RNA splice variants
- Optimization of PCR independent of RT
- Analysis of the expression of multiple genes in individual RNA samples with
oligo dT primers
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. RT-PCR may be carried out in several ways. One approach, often termed two-step RT-PCR, involves carrying out the RT (or cDNA synthesis) step in one tube and the PCR step in another tube(1) with gene specific primers, for detection of a single target from a single RNA sample. Another approach involves using one RT reaction, primed with oligo dT, for detection of multiple targets from the same RNA sample. A third approach, often termed one-step RT-PCR involves setting up the RT and PCR simultaneously with gene specific primers and carrying out the reactions sequentially in one tube(2). This is particularly useful for detection of one or a few targets in multiple RNA samples. The USB Two-Step RT-PCR Kit has been designed for use in all of these types of RT-PCR. |
All in One RT-PCR Kit
The Two-Step RT-PCR Kit comes complete and ready to use for diverse RT-PCR applications. The kit can be used with RNA samples and custom primers. The kit uses M-MLV Reverse Transcriptase(3) and Taq DNA Polymerase(4), provided in individual tubes at concentrations optimized to balance sensitivity and specificity in two-step RT-PCR. Optimized RT and PCR reaction buffers, RNase Inhibitor, Ultrapure dNTPs, supplemental magnesium chloride and RNase-free water are also included, allowing flexibility in setting up RT and PCR individually. A simple enzyme dilution step also allows use of the kit for one-step RT-PCR.
Highly Sensitive, Highly Specific
The kit can be used for detection of diverse RNA targets based on generation of long cDNAs (to at least 5.6 kb) followed by amplification of short (~0.2 to 1.5 kb) PCR products (Fig. 1). Moderately and 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(5) (Fig. 3).
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| Fig. 1. Amplification of diverse RNA targets by twostep RT-PCR. Target (source): β-actin (human liver), Numb (human liver), Ubiquitin (Arabidopsis leaf), and Terminal Deoxynucleotidyl Transferase (TdT) (calf thymus). RT was carried out on 1 µg total RNA with priming by oligo dT, and PCR was conducted on 10-1 dilution of RT reaction. Gene specific primers were designed to generate products of particular sizes. M: Marker; Lanes 1-3: β-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; Lane 8: Clathrin, 5.6 kb. |
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| Fig. 2. Highly sensitive detection of β-actin target from human liver total RNA and polyA RNA, by two-step RT-PCR with the RT Script Kit and USB Taq PCR Master Mix. RT was carried out in 25 μl volume on indicated amounts of RNA by use of a gene specific primer. PCR was conducted in a 25 μl volume on 1 μl aliquot of RT reaction. For PCR, primers were used at 0.8μM, a relatively high concentration, in order to achieve high sensitivity. Total RNA - M: Marker; Lane 1: 1 μg; Lane 2: 100 ng; Lane 3: 10 ng; Lane 4: 1 ng; Lane 5: 100 pg; Lane 6: 10 pg. PolyA RNA – M: Marker; Lane 1: 100 ng; Lane 2: 10 ng; Lane 3: 1 ng; Lane 4: 100 pg; Lane 5: 10 pg; Lane 6: 1 pg. |
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| Fig. 3. Specific detection of β-actin and Notch3 from human liver total RNA (100 ng), by two-step RT-PCR. (a) For many targets, such as 1.5 kb β-actin, specificity may be improved by decreasing the primer concentration in the PCR step. Compare results for 0.4μM versus 0.075μM primer. M: Marker; Lane 1: 0.4μM; Lane 2: 0.2μM; Lane 3: 0.1μM; Lane 4: 0.075μM. (b) For targets with high G+C contents, such as 0.34 kb Notch3 (G+C: 77%), increasing the temperature of the reverse transcription step and/or adding supplements for the RT and/or PCR steps, may improve specificity. RT reaction was carried out at 50°C without supplements, and PCR was conducted with no supplements or with 1.0M betaine. The presence of betaine results in generation of desired product. M: Marker; Lane 1: No supplement; Lane 2: 1.0M Betaine. |
Flexible Format
A wide range of RT-PCR experiments are accessible due to the all-in-one nature of this kit. One set of reagents can be applied easily in multiple types of RT-PCR, allowing great flexibility in planning, carrying out and optimizing experiments.
One Kit, Many Applications
The Two-Step RT-PCR Kit is well suited for diverse RT-PCR applications. The two-step/gene specific primer format is excellent for qualitative analysis of gene expression, highly detailed analysis of RNA splice variants, and optimization of PCR independent of RT. The
two-step/oligo dT primer format is excellent for analysis of the expression of multiple genes in individual RNA samples. And the one-step format is excellent for rapid analysis of single genes in multiple RNA samples. Depending on the scale of experiments and the size of PCR products, use of the One-Step RT-PCR Kit or the RT Script Kit coupled with the Taq PCR Master Mix or Taq PCR Kit may be more efficient. The Two-Step RT-PCR Kit is one kit with many applications.
Kit Components:
M-MLV Reverse Transcriptase
Taq DNA Polymerase
RT Reaction Buffer (5X) (includes MgCl2)
PCR Reaction Buffer (10X) (includes MgCl2)
PCR Nucleotide Mix, Ultrapure: 10mM each dATP, dCTP, dGTP, dTTP
RNase Inhibitor, Recombinant (4 units/µl)
Magnesium Chloride (25mM)
RNase-Free (DEPC-treated) Water
References:
- 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.
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