In RT-PCR, an RNA population is converted to cDNA by reverse transcription (RT), and then the cDNA is amplified by the polymerase chain reaction (PCR). Common applications of RT-PCR include detection of expressed genes, examination of transcript variants, and generation of cDNA templates for cloning and sequencing. Since reverse transcription provides cDNA templates for PCR amplification and downstream experiments, it is one of the most critical steps for experimental success.

One-step vs Two Step RT-PCR–which one is right for your experiment?

How to get high specificity one-step RT-PCR results?

Step 1. Determine RT-PCR approach

In performing RT-PCR, one-step and two-step methods are the two common approaches, each with its own advantages and disadvantages. One-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube. However, two-step RT-PCR entails two separate reactions, beginning with first-strand cDNA synthesis (RT), followed by amplification of a portion of the resulting cDNA by PCR in a separate tube.

One-step vs. two-step RT-PCR
Click image to enlarge
Figure 1. One-step vs. two-step RT-PCR.
 

One-step RT-PCR

Two-step RT-PCR

Setup Combine reaction under conditions that support both reverse transcription and PCR Separate optimized reaction for reverse transcription and PCR
Primers Gene-specific Oligo (dT), random hexamers or gene-specific primers
Ideal use Analysis of one or two genes, high-throughput platforms Analysis of multiple genes
Advantage
  • Fast
  • High-throughput
  • Fewer pipetting steps
  • Convenient
  • Reduced contamination
  • Flexible

For a faster and simpler workflow, a one-step approach is often preferred over the separate two steps.

Product highlights

Step 2. Prepare sample

Total RNA is routinely used in one-step RT-PCR. Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. The main goals of isolation workflows are to stabilize RNA molecules, inhibiting RNases, and maximizing yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity, and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Inhibitors in RT reactions

The SuperScript IV One-Step RT-PCR System is able to withstand common inhibitors of reverse transcriptase and PCR, such as co-purified compounds from biological samples or reagents used for RNA purification. This exceptional robustness makes the system less dependent on RNA sample purity to achieve reliable results.

Resistance to inhibitors

Figure 2. Resistance to inhibitors. Detection of a 1 kb RNA target from total HeLa RNA using the SuperScript IV One-Step RT-PCR System or other one-step kits in reaction mixtures containing: 1—no inhibitor, 2—heparin (0.18 μg/μL), 3—xylan (2.5 μg/μL), 4—humic acid (0.02 μg/μL), or 5—LiCl (2 μg/μL). The enzymes in all kits except the SuperScript IV One- Step RT-PCR System were inhibited by the indicated amounts of inhibitors.


Troubleshooting tips

  1. Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  2. Store RNA in an EDTA-buffered solution (0.1 mM EDTA, or 10 mM Tris + 1 mM EDTA) to minimize nonspecific cleavage by nucleases that have metal ion cofactors.

Step 3. Design primers

Superscript IV one-step RT-PCR recommends using gene-specific primers (GSP). Oligo (dT) or random primers are not recommended, because non-specific products can be generated, thereby reducing the amount of target RT-PCR products. Designing good GSP is the first step toward success of a one-step RT-PCR experiment.

Primer Design Tips

  • Design primers that anneal to the mRNA sequence in exons on both sides of an intron or exon/exon boundary, to allow differentiation between the amplified cDNA and potential contaminating genomic DNA.
  • If this approach is not feasible, consider using the next step – genomic DNA removal with ezDNase
  • Ensure primers are not self- complementary or are not complementary to each other at the 3’ end.
  • A final concentration of 0.5 uM for each primer is recommended, but further optimization may be necessary.
  • To calculate primer Tm and estimate appropriate annealing temperatures for PCR, use the Tm calculator.

Tm Calculator

The calculator calculates recommended Tm (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient. Click how to use the Tm calculator to learn more.

Step 4. Remove genomic DNA

Three common solutions are used to minimize the impact of genomic DNA in a reaction.

  • Design primers across exon-exon junctions to prevent genomic DNA amplification
  • Use control reactions without reverse transcriptase to monitor genomic DNA contamination
  • Remove genomic DNA by DNase treatment

However, genomic DNA removal with conventional DNases may cause different levels of RNA loss or damage, leading to inconsistent results. A new generation DNase Enzyme - Invitrogen ezDNase Enzyme significantly reduce the possibility of cDNA synthesis being compromised due to DNase treatment.

 ezDNase Enzyme is a recombinant double-strand-specific DNase for the fast removal of contaminating genomic DNA from RNA preparations with features include:

  •  Efficient and fast genomic DNA removal
  • Highly specific—no impact on RNA, cDNA, or primers in RT reactions

ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA or single-stranded DNA present in the reaction such as cDNA and primers. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55°C). These features make ezDNase a superior choice for genomic DNA removal prior to reverse transcription reactions.

gDNA removal procedures
Click image to enlarge
Figure 3. gDNA removal procedures: DNase I vs. Invitrogen ezDNase enzyme. Compared to DNase I, ezDNase enzyme offers a shorter workflow, simpler procedure, and less RNA damage. Inactivation of ezDNase enzyme prior to reverse transcription is optional since the enzyme does not cleave primers, ssRNA, or cDNA:RNA complexes.

Product highlights


Troubleshooting tips

  1. Trace amount of contaminants from RNA purification (e.g., SDS, EDTA) may inhibit DNase activities, therefore, re-precipitate the RNA with ethanol, wash the pellet with 75% ethanol, then dissolve in nuclease-free water.
  2. Select a gDNA removal protocol that has minimal impact on RNA integrity. If using ezDNase, increase treatment to 5 min incubation at 37oC.

Step 5. Perform one-step RT-PCR

The Invitrogen SuperScript IV One-Step RT-PCR System combines high-processivity SuperScript IV Reverse Transcriptase and high-fidelity Invitrogen Platinum SuperFi DNA polymerase to provide unmatched one-step RT-PCR performance.

Two-phase hot-start activation mechanism for high analytical specificity, yield, and room-temperature setup

Reaction setup

Superior performance with analytical sensitivity down to 0.01 pg of RNA

Detection from low amounts of input RNA

Figure 4. Detection from low amounts of input RNA. A 0.35 kb RNA target was detected from a broad range of HeLa total RNA inputs with the SuperScript IV One-Step RT-PCR System.

Target length up to 13.8 kb

Versatility across a broad range of target lengths

Figure 5. Versatility across a broad range of target lengths. Detection of human RNA fragments ranging from 0.2 to 13.8 kb with the SuperScript IV One-Step RT-PCR System.

High analytical specificity with yield with the shortest protocol

Amplification of long targets with high specificity in significantly shorter times

Figure 6. Amplification of long targets with high specificity in significantly shorter times. Detection of 7.8 kb target from total HeLa RNA using SuperScript IV One-Step RT-PCR System, SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity, and supplier QI, N, QU, T, R, and BL one-step RT-PCR products. Reactions were performed according to suppliers’ recommendations. Total reaction times for one-step RT-PCR are indicated in hours:minutes. The RNA target failed to amplify with products from suppliers QI and BL.

Product highlights


Troubleshooting tips

  1. If performing RT-PCR of long fragments, recommend increasing the concentration of template RNA.
  2. Annealing temperature and cycle numbers need to be determined according to gene specific primers and target lengths.


Tips

  • Efficient cDNA synthesis can be accomplished by a 10-minute incubation at 45–60°C. A 50°C incubation is recommended as a general starting point.
  • For GC-rich or structurally complex RNA templates, increasing the cDNA synthesis incubation temperatures up to 55–60°C may improve RT-PCR results.
  • Use 40 cycles of amplification for RT-PCR products ≤3 kb, and 35 cycles for RT-PCR products >3 kb.
  • For very low RNA input or rare targets, increasing the number of PCR cycles to 40 may improve results.
  • The PCR extension time varies with the size of the amplicon (recommended extension time is approximately 30 seconds per 1 kb of amplicon).

Consider 6 key considerations to choose a thermal cycler
Thermal Cycler video

Step 1. Determine RT-PCR approach

In performing RT-PCR, one-step and two-step methods are the two common approaches, each with its own advantages and disadvantages. One-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube. However, two-step RT-PCR entails two separate reactions, beginning with first-strand cDNA synthesis (RT), followed by amplification of a portion of the resulting cDNA by PCR in a separate tube.

One-step vs. two-step RT-PCR
Click image to enlarge
Figure 1. One-step vs. two-step RT-PCR.
 

One-step RT-PCR

Two-step RT-PCR

Setup Combine reaction under conditions that support both reverse transcription and PCR Separate optimized reaction for reverse transcription and PCR
Primers Gene-specific Oligo (dT), random hexamers or gene-specific primers
Ideal use Analysis of one or two genes, high-throughput platforms Analysis of multiple genes
Advantage
  • Fast
  • High-throughput
  • Fewer pipetting steps
  • Convenient
  • Reduced contamination
  • Flexible

For a faster and simpler workflow, a one-step approach is often preferred over the separate two steps.

Product highlights

Step 2. Prepare sample

Total RNA is routinely used in one-step RT-PCR. Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. The main goals of isolation workflows are to stabilize RNA molecules, inhibiting RNases, and maximizing yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity, and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.

Inhibitors in RT reactions

The SuperScript IV One-Step RT-PCR System is able to withstand common inhibitors of reverse transcriptase and PCR, such as co-purified compounds from biological samples or reagents used for RNA purification. This exceptional robustness makes the system less dependent on RNA sample purity to achieve reliable results.

Resistance to inhibitors

Figure 2. Resistance to inhibitors. Detection of a 1 kb RNA target from total HeLa RNA using the SuperScript IV One-Step RT-PCR System or other one-step kits in reaction mixtures containing: 1—no inhibitor, 2—heparin (0.18 μg/μL), 3—xylan (2.5 μg/μL), 4—humic acid (0.02 μg/μL), or 5—LiCl (2 μg/μL). The enzymes in all kits except the SuperScript IV One- Step RT-PCR System were inhibited by the indicated amounts of inhibitors.


Troubleshooting tips

  1. Minimize the number of freeze-thaw cycles of RNA samples to prevent degradation.
  2. Store RNA in an EDTA-buffered solution (0.1 mM EDTA, or 10 mM Tris + 1 mM EDTA) to minimize nonspecific cleavage by nucleases that have metal ion cofactors.

Step 3. Design primers

Superscript IV one-step RT-PCR recommends using gene-specific primers (GSP). Oligo (dT) or random primers are not recommended, because non-specific products can be generated, thereby reducing the amount of target RT-PCR products. Designing good GSP is the first step toward success of a one-step RT-PCR experiment.

Primer Design Tips

  • Design primers that anneal to the mRNA sequence in exons on both sides of an intron or exon/exon boundary, to allow differentiation between the amplified cDNA and potential contaminating genomic DNA.
  • If this approach is not feasible, consider using the next step – genomic DNA removal with ezDNase
  • Ensure primers are not self- complementary or are not complementary to each other at the 3’ end.
  • A final concentration of 0.5 uM for each primer is recommended, but further optimization may be necessary.
  • To calculate primer Tm and estimate appropriate annealing temperatures for PCR, use the Tm calculator.

Tm Calculator

The calculator calculates recommended Tm (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient. Click how to use the Tm calculator to learn more.

Step 4. Remove genomic DNA

Three common solutions are used to minimize the impact of genomic DNA in a reaction.

  • Design primers across exon-exon junctions to prevent genomic DNA amplification
  • Use control reactions without reverse transcriptase to monitor genomic DNA contamination
  • Remove genomic DNA by DNase treatment

However, genomic DNA removal with conventional DNases may cause different levels of RNA loss or damage, leading to inconsistent results. A new generation DNase Enzyme - Invitrogen ezDNase Enzyme significantly reduce the possibility of cDNA synthesis being compromised due to DNase treatment.

 ezDNase Enzyme is a recombinant double-strand-specific DNase for the fast removal of contaminating genomic DNA from RNA preparations with features include:

  •  Efficient and fast genomic DNA removal
  • Highly specific—no impact on RNA, cDNA, or primers in RT reactions

ezDNase Enzyme's high specificity for double-stranded DNA enables efficient and fast genomic DNA removal without reduction in the quality or quantity of RNA or single-stranded DNA present in the reaction such as cDNA and primers. ezDNase Enzyme is heat-labile and so can be easily deactivated by heat treatment at moderate temperature (55°C). These features make ezDNase a superior choice for genomic DNA removal prior to reverse transcription reactions.

gDNA removal procedures
Click image to enlarge
Figure 3. gDNA removal procedures: DNase I vs. Invitrogen ezDNase enzyme. Compared to DNase I, ezDNase enzyme offers a shorter workflow, simpler procedure, and less RNA damage. Inactivation of ezDNase enzyme prior to reverse transcription is optional since the enzyme does not cleave primers, ssRNA, or cDNA:RNA complexes.

Product highlights


Troubleshooting tips

  1. Trace amount of contaminants from RNA purification (e.g., SDS, EDTA) may inhibit DNase activities, therefore, re-precipitate the RNA with ethanol, wash the pellet with 75% ethanol, then dissolve in nuclease-free water.
  2. Select a gDNA removal protocol that has minimal impact on RNA integrity. If using ezDNase, increase treatment to 5 min incubation at 37oC.

Step 5. Perform one-step RT-PCR

The Invitrogen SuperScript IV One-Step RT-PCR System combines high-processivity SuperScript IV Reverse Transcriptase and high-fidelity Invitrogen Platinum SuperFi DNA polymerase to provide unmatched one-step RT-PCR performance.

Two-phase hot-start activation mechanism for high analytical specificity, yield, and room-temperature setup

Reaction setup

Superior performance with analytical sensitivity down to 0.01 pg of RNA

Detection from low amounts of input RNA

Figure 4. Detection from low amounts of input RNA. A 0.35 kb RNA target was detected from a broad range of HeLa total RNA inputs with the SuperScript IV One-Step RT-PCR System.

Target length up to 13.8 kb

Versatility across a broad range of target lengths

Figure 5. Versatility across a broad range of target lengths. Detection of human RNA fragments ranging from 0.2 to 13.8 kb with the SuperScript IV One-Step RT-PCR System.

High analytical specificity with yield with the shortest protocol

Amplification of long targets with high specificity in significantly shorter times

Figure 6. Amplification of long targets with high specificity in significantly shorter times. Detection of 7.8 kb target from total HeLa RNA using SuperScript IV One-Step RT-PCR System, SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity, and supplier QI, N, QU, T, R, and BL one-step RT-PCR products. Reactions were performed according to suppliers’ recommendations. Total reaction times for one-step RT-PCR are indicated in hours:minutes. The RNA target failed to amplify with products from suppliers QI and BL.

Product highlights


Troubleshooting tips

  1. If performing RT-PCR of long fragments, recommend increasing the concentration of template RNA.
  2. Annealing temperature and cycle numbers need to be determined according to gene specific primers and target lengths.


Tips

  • Efficient cDNA synthesis can be accomplished by a 10-minute incubation at 45–60°C. A 50°C incubation is recommended as a general starting point.
  • For GC-rich or structurally complex RNA templates, increasing the cDNA synthesis incubation temperatures up to 55–60°C may improve RT-PCR results.
  • Use 40 cycles of amplification for RT-PCR products ≤3 kb, and 35 cycles for RT-PCR products >3 kb.
  • For very low RNA input or rare targets, increasing the number of PCR cycles to 40 may improve results.
  • The PCR extension time varies with the size of the amplicon (recommended extension time is approximately 30 seconds per 1 kb of amplicon).

Consider 6 key considerations to choose a thermal cycler
Thermal Cycler video

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