Using ImProm-II™ Reverse Transcription System for Two-Step RT-PCR

Introduction

The ImProm-II™ Reverse Transcription System (Cat.# A3800) is a convenient kit that combines a newly formulated reverse transcriptase, an optimized reaction buffer and the associated reagents qualified for efficient synthesis of first-strand cDNA. The system is designed for use in synthesizing high-quality cDNA in preparation for gene-specific analysis such as PCR amplification. Moreover, use of the ImProm-II™ Reverse Transcription System allows experimental questions to be addressed and optimized as two separate steps (two-step RT-PCR). The components of the ImProm-II™ Reverse Transcription System are provided separately to allow users to individualize and optimize protocols.

Two-step RT-PCR assays demand flexibility. The volume of the RT reaction product that is applied to each amplification and the final PCR volumes vary from assay to assay. In some cases, a single cDNA synthesis reaction may be analyzed for a single expressed gene in a very small reaction volume. In other cases, numerous expression analyses may be desired from one cDNA synthesis reaction. Some assays require that amplification mixes are prepared using individual components to allow for individual assay concentration requirements. In other situations, the PCR Master Mix (Cat.# M7501), with its standardized composition, is optimal. When this flexibility is required, the ImProm-II™ Reverse Transcription System provides for robust application to RT-PCR assays when linked with amplification using Promega products for PCR.

In this article, we illustrate the flexibility and range of efficient analysis enabled through the use of the ImProm-II™ Reverse Transcription System for two-step RT-PCR.

Results

Varying the Amount of RT Reaction Used in the PCR Step: Anywhere from 1µl to the entire 20µl of the cDNA synthesis reaction may be transferred directly into 100µl amplification reactions without inhibiting PCR. Figure 1 shows that we detected as few as 10 copies of a transcript using the ImProm-II™ Reverse Transcription System linked in 100µl, gene-specific, two-step reactions using Taq DNA Polymerase. Reverse Transcription reactions containing titrated amounts of poly(A)+ kanamycin RNA primed with Oligo(dT)₁₅ were subjected to PCR specific for the 3´-end of the 1.2kb kanamycin cDNA, demonstrating that the RT reaction made full-length DNA.

Figure 1. Sensitivity of ImProm-II™ Reverse Transcriptase reactions in two-step RT-PCR.

Poly(A)+ kanamycin transcript RNA was titrated over a range of approximately 10¹⁰ (0.1µg) to 10¹ copies, including a no-RNA negative control reaction. Each RT reaction was scaled up to provide enough cDNA for several applications. The RNA template was primed with oligo(dT)₁₅ for standard cDNA synthesis. The RT products (1µl in Panel A or 20µl in Panel B) were subsequently applied directly into 100µl amplification reactions (Methods D and E) for kanamycin-specific PCR amplification of the terminal 3´ sequences of the cDNA. PCR profile: 38 cycles of (94°C for 1 minute; 60°C for 1 minute; 72°C for 2 minutes) followed by final extension at 72°C for 5 minutes. Samples (10µl) of each RT-PCR were analyzed for the presence of the 323bp product on a 4% NuSieve®:GTG® agarose TBE gel.  Lane M, 100bp DNA Ladder (Cat.# G2101).

Using Individual Reagents or PCR Master Mix for PCR: In two-step RT-PCR, ImProm-II™ Reverse Transcription reaction products may be subjected to gene-specific amplification reactions that have been prepared using individual components or reactions containing Promega PCR Master Mix. Oligo(dT)₁₅-primed cDNA was synthesized in reactions containing titrated amounts of human Jurkat total RNA; 20µl of the reverse transcription reaction products were transferred into 50µl amplification reactions specific for the caspase-3 message. Figure 2 demonstrates that two-step RT-PCR using ImProm-II™ reagents linked with PCR Master Mix produces results that are equivalent to results obtained using ImProm-II™ reagents and standalone PCR components.

Figure 2. Individual reaction components (Panel A) or PCR Master Mix (Panel B) yield comparable results in two-step RT-PCR.

In the same RT reactions described in Figure 3, oligo(dT)₁₅-primed cDNA was synthesized using Jurkat total RNA over the indicated range of concentrations. A 20µl aliquot of each reaction was then applied to caspase-3 gene-specific amplifications (50µl). The amplification mixes were prepared using either standalone Promega PCR reagents (Method B, Panel A) or Promega PCR Master Mix (Method C, Panel B). PCR profile: 40 cycles of (94°C for 1 minute; 60°C for 1 minute; 72°C for 2 minutes) followed by final extension at 72°C for 5 minutes. Samples (5µl) of each RT-PCR were analyzed for the presence of the appropriately sized 533bp product on a 4% NuSieve®:GTG® agarose TBE gel. Lane M, 100bp DNA Ladder (Cat.# G2101).

Amplifying Different Transcripts from One RT Reaction: We demonstrated the ability to amplify messages of differing abundance by synthesizing a single batch of cDNA using total RNA primed with Oligo(dT)₁₅ and then dividing the resulting cDNA into different gene-specific amplification reactions. We used total RNA isolated from cultured human Jurkat cells as template for ImProm-II™ RT reactions. Aliquots (20µl) of the cDNA product of the reverse transcription reactions were transferred into separate 50µl amplification reactions specific for several different mRNA target sequences of varied abundance ⁽¹⁾ : high-abundance, β-actin ⁽²⁾ ; medium-abundance, γ-actin ⁽³⁾ ; low-abundance, Adenosine Ribosylation Factor 1 (ARF-1); and presumably low abundance caspase-3. The different transcripts were detected in reactions containing a range of RNA template concentrations from 100ng down to 1pg of total RNA (Figure 3).

Figure 3. Amplification of several different transcripts of differing abundance from one RT reaction.

To demonstrate the sensitivity of reactions for several different mRNA target sequences of varied abundance in a complex population of RNAs, total RNA was isolated from cultured Jurkat cells using Promega SV Total RNA Isolation System. Batches of cDNA were synthesized from oligo(dT)₁₅-primed total RNA from 100ng to 1pg of total RNA per 20µl RT reaction volume. Each RT reaction was scaled up to provide enough volume to afford 20µl of cDNA for several different gene-specific amplifications (Method B). The 50µl amplification reactions were designed to detect relative expression of four different genes: β-actin (highly abundant), γ-actin (moderately abundant), ARF-1 (low abundance) as well as human caspase-3. PCR profile: 40 cycles of 94°C for 1 minute; 60°C for 1 minute; 72°C for 2 minutes followed by final extension at 72°C for 5 minutes. Samples (5µl) of each amplification were analyzed for the presence of the appropriately sized product on a 4% NuSieve®:GTG® agarose TBE gel. Lane M, 100bp DNA Ladder (Cat.# G2101).

Adding Taq DNA Polymerase and Primers Directly to the RT Reaction: For ultimate sensitivity and efficiency, the separate steps of two-step RT-PCR may be performed by supplementing the original 20µl RT reaction volume with PCR reagents. The Improm-II™ RT Buffer supports PCR in addition to reverse transcription. Following oligo(dT)₁₅-primed ImProm-II™ RT reactions, we supplemented the 20µl reaction directly with Taq DNA Polymerase and gene-specific primers. As shown in Figure 4, cDNA synthesized from Jurkat total RNA (between 100ng and 10pg per 20µl RT reaction) was supplemented with 1µl of Taq DNA Polymerase and 20pmol of each gene-specific primer. This method enhances efficiency and provides the potential for "hot start" addition of the Taq DNA Polymerase.

Figure 4. Two-step RT-PCR with simple addition of Taq DNA Polymerase and gene-specific primers to the original 20µl RT volume.

In the same reverse transcriptase reactions described in Figure 3, oligo(dT)₁₅-primed cDNA was synthesized using Jurkat total RNA over the indicated range of concentrations (Method A). While the inactivated reverse transcriptase reactions were warming in the thermal cycler at 94°C for the initial PCR denaturation step, we performed a manual "hot start" addition of 1µl of Taq DNA polymerase and 1µl of gene-specific primers to each 20µl reaction for caspase-3-specific amplifications (Panel A). This method was run in parallel with the two-step amplification of Method B consisting of the addition of 20µl of RT product to 30µl of PCR mix (Panel B). PCR profile: 40 cycles of (94°C for 1 minute; 60°C for 1 minute; 72°C for 2 minutes) followed by final extension at 72°C for 5 minutes. Samples (5µl) of each RT-PCR were analyzed for the presence of the appropriately sized product on a 4% NuSieve:GTG agarose TBE gel. Lane M, 100bp DNA Ladder.

Performing Full-Length cDNA Synthesis of a Large Transcript: Full-length cDNA (up to 8.9kb) can be synthesized using the ImProm-II™ Reverse Transcription System as demonstrated by two-step RT-PCR specific for the APC (adenomatous polyposis coli) message. HeLa total RNA (1µg) was used as template for cDNA synthesis using either Oligo(dT)₁₅, random hexamers or an APC primer specific to the 3´ end of the APC mRNA. The APC message is challenging to amplify because it is 8.9kb in length, and it is expressed at relatively low abundance in HeLa cells. Aliquots (20µl or 1µl) of the reverse transcription reactions were amplified in 100µl volume PCR specific for the 3´ sequences of the cDNA. As demonstrated in Figure 5, the ImProm-II™ Reverse Transcription System produced the correct size of amplification product specific for the 3´-end of this long transcript using only 1µl of the RT reaction for PCR.

Figure 5. Full-length cDNA synthesis using three different primers in two-step RT-PCR.

HeLa total RNA (1µg/reaction), and a no-RNA negative control reaction, were reverse transcribed using either oligo(dT)₁₅, random hexamers or an APC gene-specific primer targeted to the 3´ end of the APC mRNA. The APC message is 8.9kb in length and is expressed in HeLa cells at relatively low abundance. One or 20µl of the reverse transcription products were then amplified according to Methods C and D. PCR profile: 38 cycles of (94°C for 1 minute; 60°C for 1 minute; 72°C for 3 minutes) followed by final extension at 72°C for 5 minutes and soak at 4°C. Samples (5µl) of the reaction products were analyzed on a 2% Latitude® agarose gel. Lanes 1, Oligo(dT)₁₅ Primer; lanes 2, random primers; lanes 3, gene-specific primer. Lane M, 100bp DNA Ladder.

Conclusions

For two-step RT-PCR applications, the ImProm-II™ Reverse Transcription System can be used for first-strand cDNA synthesis over a range of RNA template concentrations. The system can process from 1µg to less than 1pg of total RNA, depending on the abundance of the message. The ImProm-II™ RT reactions can be initiated using a variety of cDNA primers, including Oligo(dT)₁₅, Random Hexamers and gene-specific primers. Because the ImProm-II™ Reverse Transcription buffer supports RT and PCR and has the added benefit of suppressing any inhibition of Taq DNA Polymerase activity, the amount of the reverse transcription reaction products that can be transferred into PCR, and the volumes of the amplification reactions can be varied based upon experimental design. Between 1µl and 20µl of the reverse transcription reaction products including PCR Master Mix can be added directly to PCR using Promega reagents for amplification. The amplifications may be performed in volumes between 20µl and 100µl. Full-length cDNA synthesis up to 8.9kb and excellent sensitivity are observed with a variety of templates when ImProm-II™ reverse transcription is linked to gene-specific amplifications using a variety of formats.

Methods

The SV Total RNA Isolation System (Cat.# Z3100) and the supplied protocol (Technical Manual #TM048) were used to isolate total RNA from human Jurkat cells or human HeLa cell cultures. Kanamycin Positive Control RNA (1.2kb, Cat.# C1381) was used as a single-species, poly(A)+ RNA transcript for template titrations of a single target.

For all RNA titrations, RNA was serially diluted into ice-cold, nuclease-free water. For each 20µl of RT-PCR, we combined aliquots containing known amounts of diluted RNA with primers in thin-walled, nuclease-free tubes on ice. We denatured samples at 70°C for 5 minutes, chilled on ice and then added 5µl of the RNA + primer mix to each reaction. RNA-primer combinations as outlined in Table 1 were used to illustrate the success of RT-PCR using gene-specific primers alone or in combination with Oligo(dT)₁₅ primer. The no-template, negative control reactions consisted of water and primer but no RNA. Table 1 shows the sequences of the gene-specific primers used in the amplifications and the expected product sizes generated by amplification of the cDNA.

Table 1. Target RNA and Gene-Specific Oligonucleotide Primers.

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Notes: Kanamycin Positive Control RNA: 0.1µg, 2.5fmol (~10¹⁰ copies) to 25ymol (~10 copies).

For APC mRNA target, between 10ng and 1µg of Jurkat cell total RNA were used; for all other mRNA, between 1pg and 100ng were used.

First-Strand cDNA Synthesis for Two-Step RT-PCR Methods: Human total RNA or kanamycin poly(A)+ transcript RNA were used as template for cDNA synthesis by oligo(dT)₁₅ priming. For each RT reaction (20µl), we combined the components of the ImProm-II™ Reverse Transcription System on ice. We included all components except RNA template + primer, and we scaled the volumes in batches to accommodate the number of tubes in the experimental examples. We transferred 15µl of RT Reaction Mix to each prepared tube of 5µl of target + primer combination and topped each reaction volume with nuclease-free mineral oil to prevent evaporation. The setup is listed in Table 2.

Table 2. RT-PCR Setup.

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Notes: MgCl₂ may be optimized to a final concentration of 1.5–8.0mM.

We transferred tubes to a programmed, controlled temperature block for coupled reverse transcription and amplification as follows: Primer and template annealing, 25°C for 5 minutes; first-strand extension, 42°C for 60 minutes; and heat inactivation, 70°C for 15 minutes. Amplification conditions are listed in the figure legends.

Two-step PCR Using Promega PCR Reagents: Either all (20µl) or part (1µl) of the oligo(dT)-primed cDNA from the RT reactions above were supplemented with PCR components for reactions of different volumes as specified. We prepared different combinations of ImProm-II™ RT reaction product volumes and PCR components as outlined below. Because of carryover of ImProm-II™ Reaction Buffer, magnesium and dNTPs; the amounts of additional buffer, magnesium and dNTPs vary in each example, to provide the optimal concentrations.

Method A: We added 1µl of Taq DNA Polymerase and 1µl of gene-specific primer mix to the entire 20µl of RT reaction product for a 22µl PCR. The addition was made to the reactions during the initial 94°C denaturation of the PCR cycles, effecting a manual "hot start" method.

Method B: We prepared 30µl of gene-specific PCR mix, vortexed gently and added it to 20µl of RT reaction product for a single 50µl PCR. 

Method C: We substituted PCR Master Mix for individual components to prepare 30µl of gene-specific PCR mix and added it to 20µl of RT reaction product for a 50µl PCR. 

Method D: We prepared 80µl of gene-specific PCR mix, mixed it and added it to the entire 20µl of RT reaction product for a single 100µl PCR.

Method E: We prepared 99µl of gene-specific PCR mix and added it to 1µl of RT reaction product.

Table 3. Five Procedures for Amplification Following First-Strand cDNA Synthesis.

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The reactions were overlaid with mineral oil to prevent evaporation. The reactions were placed in a programmed thermal cycler and incubated for PCR as specified in the examples.

Note: For additional information on optimizing first-strand synthesis, see the Technical Manual for the ImProm-II™ Reverse Transcription System ( #TM236).