Description

The ThermoScript™ RT-PCR System is designed for the sensitive and reproducible detection and analysis of RNA molecules in a two-step process. ThermoScript™ RT, an avian reverse transcriptase with reduced RNase H activity, is engineered to have higher thermal stability, produce higher yields of cDNA, and produce more full-length cDNA transcripts than AMV RT. cDNA synthesis is performed in the first step using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers or a gene-specific primer, at 50-65°C. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest. RNA targets from 100 bp to >12 kb can be detected with this system, using 10 pg to 5 Yg of total RNA. PCR is carried out with Platinum® Taq DNA Polymerase or Platinum® Taq DNA Polymerase High Fidelity. Platinum® Taq DNA Polymerase High Fidelity is suitable for templates from 100 bp to >12 kb. Platinum® Taq DNA polymerase (1) provides automatic hot-start conditions for increased specificity up to 3 kb.

Reagents are provided for 25 or 100 cDNA synthesis reactions of 20 μl each and 25 or 100 amplification reactions of 50 μl each.

Component 25 rxn kit 100 rxn kit
ThermoScript™ RT (15 U/Yl)25 μl100 μl
5X cDNA Synthesis Buffer*500 μl500 μl
0.1 M DTT250 μl250 μl
10 mM dNTP Mix100 μl2 × 250 μl
RNaseOUT™ (40 U/Yl)25 μl100 μl
Oligo (dT)20 (50 YM)25 μl100 μl
Random Hexamers (50 ng/Yl)50 μl250 μl
DEPC-Treated Water1.25 ml1.25 ml
E. coli RNase H (2 U/Yl)50 μl2 × 50 μl

*250 mM Tris acetate (pH 8.4), 375 mM potassium acetate, 40 mM magnesium acetate, stabilizer

Catalog numbers 11146-057 (25 rxns) and 11146-032 (100 rxns) include the following, in addition to the components to the left:

Component 25 rxn kit 100 rxn kit
Platinum® Taq DNA polymerase (5 U/Yl)100 units250 units
10X PCR buffer Minus Mg1.0 ml1.0 ml
50 mM MgCl21.0 ml1.0 ml


Catalog number 11146-040 (100 rxns) includes the following, in addition to the components to the left:

Component 100 rxn kit
Platinum® Taq DNA Polymerase
High Fidelity (5 U/μl)
100 units
10X High Fidelity PCR Buffer1.0 ml
50 mM MgSO41.0 ml

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Summary of Procedure

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Important Parameters to Consider

RNA
  • High quality intact RNA is essential for successful full-length cDNA synthesis and successful long RTPCR.
  • RNA should be devoid of any RNase contamination and aseptic conditions should be maintained.
  • Recommended methods of total RNA isolation include the Micro-to-Midi Total RNA Purification System (Catalog no. 12183-018) and TRIzol® Reagent (Catalog no. 15596-026) (2, 3). Oligo(dT)-selection for poly(A)+ RNA is typically not necessary, although incorporating this step may improve the yield of specific cDNAs.

cDNA Synthesis Primers


  • Oligo(dT)20 (50 pmoles/reaction) is recommended for priming polyadenylated RNA. Use of Oligo(dT)20 allows the detection of multiple transcripts from a single first-strand reaction.
  • Random hexamers (50-250 ng/reaction) are efficient primers for the detection of multiple short RT-PCR targets. Use of more than 50-100 ng primer/Yg of RNA can increase the yield of short products but may inhibit detection of long targets (>3kb) or rare transcripts. If random hexamers are used, the firststrand reaction must be incubated at 25°C for 10 min to extend the primers prior to increasing the reaction temperature for synthesis.
  • Gene-specific primers (GSP) should be used at 10 to 20 pmol/reaction. Specificity of priming may be improved by optimizing annealing/reaction temperature.
  • Treatment of cDNA with RNase H to remove the complementary RNA prior to PCR is optional. RNase H digestion will improve the RT-PCR signal of many targets and is required for the efficient and consistent amplification of long RT-PCR templates. 


cDNA Synthesis Reaction

  • Denaturation of the RNA template and primer by incubating at 65°C for 5 min is optional. Most targets can be reverse transcribed efficiently without this step. However, heating the RNA in the absence of reaction buffer and enzyme prior to cDNA synthesis can remove secondary structure that may impede full length cDNA synthesis.
  • ThermoScript™ RT can be used at 50-65°C. We recommend incubation at 50-55°C for most RT-PCR targets. However, incubation at 50-60°C for oligo(dT) and 50-65°C for gene-specific primers can be employed to reduce secondary structure or to improve specificity.
  • Most targets can be amplified after only a 30-min incubation for the first-strand reaction. Rare RNAs, long transcripts, or targets at the 5′ end of long transcripts benefit from longer incubation times (50-60 min).

PCR Primers

  • A final primer concentration of 0.2 - 0.4 μM for each primer is generally optimal; however, a primer titration is recommended for best results.
  • Design primers that anneal to sequence in exons on both sides of an intron or exon/exon boundary of the mRNA to allow differentiation between amplification of cDNA and potential contaminating genomic DNA.
  • Primers should not be self-complementary or complementary to each other at the 3′ ends. 

PCR Reactions
  • Most targets will be efficiently amplified using 2 Yl or less of the cDNA synthesis reaction.
  • The optimum magnesium concentration varies from 1.5 to 3 mM. Generally, 1.82 mM magnesium chloride for Platinum® Taq DNA polymerase and 2.32 mM magnesium sulfate for Platinum® Taq DNA Polymerase High Fidelity is effective for most primer sets. However, titration of the magnesium concentration is recommended for the best result. Each Yl of the cDNA synthesis reaction adds 0.16 mM to the final magnesium concentration in a 50-μl PCR reaction.
  • Assemble the PCR reactions on ice, transfer them to a pre-heated thermal cycler (85-95°C) and immediately start the PCR amplification program.
  • The annealing temperature should be 10°C below the melting temperature of the primers used.
  • The optimum extension temperature for Platinum® Taq DNA Polymerase High Fidelity is 68°C. The extension time varies with the size of the amplicon (approximately 1 min per 1 kb of amplicon).

cDNA Synthesis

  1. In a 0.2- or 0.5-ml tube, combine primer (50 μM Oligo(dT)20, 50 ng/μl random primer or 10 μM gene-specific primer), RNA, and dNTP mix and adjust volume to 12 Yl with DEPC-treated water.

    Component Amount
    Primer1 yl
    RNA (10 pg -5 Yg)x yl
    10 mM dNTP Mix2 yl
    DEPC-treated waterto 12 yl


  2. Denature RNA and primer by incubating at 65°C for 5 min and then place on ice (optional).

  3. Vortex the 5X cDNA Synthesis Buffer for 5 s just prior to use.

  4. Prepare a master reaction mix on ice and vortex gently.



    Component 1 Reaction 10 Reactions
    Primer1 Yl
    RNA (10 pg -5 Yg)RNA (10 pg -5 Yg)
    10 mM dNTP Mix2 Yl
    DEPC-treated waterto 12 Yl

    2. Denature RNA and primer by incubating at 65°C for 5 min and then place on ice (optional).

    3. Vortex the 5X cDNA Synthesis Buffer for 5 s just prior to use.

    4. Prepare a master reaction mix on ice and vortex gently.

    *Note: If less than 1 ng of template RNA is used, reduce the amount of ThermoScript™ RT in the reaction to 0.5 μl/reaction (5 μl/10 reactions). Increase the amount of DEPC-treated water in the master reaction mix to 1.5 μl/reaction (15 μl/10 reactions).

    5. Pipet 8 Yl of master reaction mix into each reaction tube on ice.

    6. Transfer the sample to a thermal cycler preheated to the appropriate cDNA synthesis temperature and incubate as follows. Oligo(dT)20 primed: 30-60 min at 50°C (or 50-60°C) Gene-specific primed 30-60 min at 50°C (or 50-65°C) Random-hexamer primed: 25°C for 10 min, followed by 20- 50 min at 50°C (or 50-65°C)

    7. Terminate the reaction by incubating at 85°C for 5 min.

    8. Add 1 Yl of RNase H and incubate at 37°C for 20 min (optional).

    9. cDNA synthesis reactions can be stored at -20°C or used for PCR immediately.

    PCR with Platinum® Taq DNA Polymerase High Fidelity

    Use only 10% of the cDNA synthesis reaction (2 Yl) for PCR. Use of 2 μl of 50 mM MgSO4 and 2 Yl of cDNA (0.32 mM magnesium in a 50-Yl PCR) results in a final concentration of 2.32 mM magnesium, which is effective for most primer sets. However, titration of the magnesium concentration with the provided 50 mM MgSO4 is recommended for best results.

    1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube:


    Component 1 Reaction 10 Reactions
    10X High Fidelity PCR Buffer5 Yl50 Yl
    50 mM MgSO42 μl20 μl
    10 mM dNTP Mix1 Yl10 Yl
    10 YM sense primer
    1 Yl
    10 Yl
    10 YM antisense primer1 Yl10 Yl
    Platinum® Taq High Fidelity
    0.2 Yl
    2 Yl
    cDNA (from cDNA synthesis reaction)
    2 Yl
    20 Yl
    DEPC-treated water
    37.8 Yl
    378 Yl
    Final volume
    50 Yl
    500 μl



    2. Mix gently and overlay with silicone oil or mineral oil if the thermal cycler lacks a heated lid.

    3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of PCR with optimized conditions for your sample (1 min/kb extension time at 68°C).

    4. Analyze 10 Yl of the amplified sample by agarose gel electrophoresis.

    PCR with Platinum® Taq DNA Polymerase

    Use only 10% of the cDNA synthesis reaction (2 Yl) for PCR. Use of 50 mM MgCl2 and 2 Yl of cDNA will result in a final magnesium concentration of 1.82 mM, which is adequate for most primers and targets. However, titration of magnesium concentration is recommended for best results.

    1. Add the following to a 0.2- or 0.5-ml, thin-walled, PCR tube:
     


    Component 1 Reaction 10 Reactions
    10X PCR Buffer Minus Mg 5 Yl50 Yl
    50 mM MgCl21.5 Yl15 Yl
    10 mM dNTP Mix1 Yl
    10 Yl
    10 YM sense primer
    1 Yl
    10 Yl
    10 YM antisense primer1 Yl10 Yl
    Platinum® Taq DNA polymerase (5 U /Yl)
    0.4 Yl
    4 Yl
    cDNA (from cDNA synthesis reaction)
    2 Yl
    20 Yl
    DEPC-treated water
    38.1 Yl
    381 Yl
    Final volume
    50 Yl
    500 μl

    2. Mix gently and overlay with silicone oil or mineral oil if the thermal cycler lacks a heated lid.
    3. Incubate at 94°C for 2 min, then perform 20 to 40 cycles of PCR with optimized conditions for your sample (1 min/kb extension time at 68-72°C)
    4. Analyze 10 Yl of the amplified sample by agarose gel electrophoresis.

    Control Reactions

    An RT-PCR Primer and Control Set is available separately for monitoring the performance of the system (Cat. Number 10929-016).
    1. Use 1 ng of the Control RNA in the cDNA Synthesis Reaction.
    2. Perform the PCR using Platinum® Taq DNA Polymerase, as described above.
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Troubleshooting

RT-PCR Problem Possible Cause Suggested Solution
Little or no RT-PCR product visible after agarose gel analysis. 
RNA was degraded. 
Analyze RNA on a denaturing gel before use to verify integrity. Use aseptic technique for RNA isolation. Process tissue immediately after removal from animal. Store RNA in 100% formamide. If using placental RNase inhibitor, do not heat >45°C or use >pH 8.0 or inhibitor may release any bound RNases. Also, DTT must be present when the RNase inhibitor is added at =0.8 mM DTT. 
Little or no RT-PCR product visible after agarose gel analysis.
RNA contained an inhibitor of RT.
Remove inhibitor by ethanol precipitation of the RNA. Include a 70% (v/v) ethanol wash of the RNA pellet. Glycogen (0.25 µg to 0.4 µg/µl) can be included to aid in RNA recovery for small samples. Inhibitors of RT include: SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, and guanidinium salts. Test for inhibitors by mixing a control RNA with the sample and comparing yields to control RNA reaction. 
Little or no RT-PCR product visible after agarose gel analysis. 
Polysaccharide coprecipitation of RNA. 
Precipitate RNA with lithium chloride to remove polysaccharides.
Little or no RT-PCR product visible after agarose gel analysis. Primer used for first-strand cDNA synthesis did not anneal well. Be sure annealing temperature is appropriate for your primer. For random hexamers, a 10 min. incubation at 25°C is recommended before incubating at reaction temperature. For gene-specific primers (GSP), try another GSP or switch to oligo(dT) or random hexamers. Make sure GSP is the antisense sequence. 
Little or no RT-PCR product visible after agarose gel analysis.Not enough starting RNA. Increase the amount of RNA. For <50 ng RNA, use 0.1 µg to 0.5 µg acetylated BSA or 40 units of RNaseOUT™ Ribonuclease Inhibitor in first-strand cDNA synthesis (3,4). To maximize RT-PCR sensitivity, use the SuperScript™ III First-Strand Synthesis System for RT-PCR.   
Little or no RT-PCR product visible after agarose gel analysis. RNA template had high secondary structure. Denature/anneal RNA and primers in the absence of salts and buffer. Raise the RT reaction temperature up to 55°C for SuperScript™ III or up to 65°C for ThermoScript™ RT (5). Note: Do not use oligo(dT) as a primer over 60°C and choose a GSP that will anneal at your reaction temperature. For RT-PCR products >1 kb, keep reaction temperature = 65°C. Do not use M-MLV RT above 37°C. Use random hexamers in the first-strand reaction if full-length cDNA is not needed. 
Little or no RT-PCR product visible after agarose gel analysis The primers or template are sensitive to remaining RNA template. Treat first-strand cDNA with RNase H before PCR.
Little or no RT-PCR product visible after agarose gel analysis.  
Target not expressed in tissue analyzed. 
Try a different target or tissue. 
Little or no RT-PCR product visible after agarose gel analysis. 
PCR did not work. For two-step RT-PCR, do not use more than 1/5 of the RT reaction in the PCR step.
Little or no PCR product visible after agarose gel analysis
Poor PCR primer design.
Avoid complementary sequences at the 3´ end of primers. Avoid sequences that can form internal hairpin structures. Design primers with similar Tms.
Little or no PCR product visible after agarose gel analysis
DNA contains inhibitors.
Reagents such as DMSO, SDS, and formamide can inhibit Taq DNA polymerase. If inhibitor contamination is suspected, ethanol precipitate the DNA sample.
Little or no PCR product visible after agarose gel analysis
GC-rich template
For templates >50% GC content, use PCRx Enhancer Solution, or the Accuprime GC-Rich DNA polymerase.
Little or no PCR product visible after agarose gel analysis
Template concentration is too low.
Start with 1000 copies of the target sequence to obtain a signal in 25 to 30 cycles.
Little or no PCR product visible after agarose gel analysis
Magnesium concentration is too low.
Determine the optimal magnesium concentration for each template and primer pair by performing a reaction series from 1 mM to 3 mM in 0.5 mM increments. Note: Use 3 mM to 5 mM magnesium for real-time quantitative PCR.
Little or no PCR product visible after agarose gel analysis
Annealing temperature is too high.
Estimate the Tm using a computer program or equation and set the annealing temperature 5°C below the Tm. Since these equations estimate Tm values, the true annealing temperature may actually be higher or lower.
Little or no PCR product visible after agarose gel analysis
Primer concentration is too low.
Optimal primer concentration is between 0.1 µM to 0.5 µM. To accurately determine primer concentration, read the optical density at 260 nm (OD260). Then, calculate the concentration using the absorbance and the extinction coefficient.
Unexpected bands after gel analysis.
Non-specific annealing of primers to templates.
Use a GSP instead of random hexamers or oligo(dT) for first-strand synthesis. Try a GSP that allows cDNA synthesis at high temperatures
Unexpected bands after gel analysis.
Poor GSP design.
Follow the same rules as described for amplification primers.
Unexpected bands after gel analysis.
Genomic DNA contamination of RNA.
Treat RNA with DNase I, Amplification Grade. Check for DNA contamination with a control reaction without RT.
Unexpected bands after gel analysis.
Primer-dimer formation. 
Design primers without complementary sequences at the 3´ ends.
Unexpected bands after gel analysis. 
Non-specific annealing of primers to template.
Increase the annealing temperature in 2°C to 5°C increments and minimize the annealing time. Use higher annealing temperatures for the first few cycles, followed by lower annealing temperatures. Use Platinum® Ta q DNA Polymerase for automatic hot-start PCR (9). Avoid 2 or 3 dGs or dCs at the 3´ end of primers. 
Unexpected bands after gel analysis.
Magnesium concentration is too high.
Optimize magnesium concentration for each template and primer combination.
 Unexpected bands after gel analysis. 
Primer mispriming due to amplification from complex templates.
Use nested primers or touchdown PCR.
Unexpected bands after gel analysis.
Contaminating DNA from an exogenous source.
Use aerosol-resistant tips and UDG.
Unexpected bands after gel analysis.
Primer binding sites are inaccessible due to secondary structure. 
For templates >50% GC content use (1X-3X) PCRx Enhancer Solution, Accuprime GC-Rich DNA Polymerase, or additives.
PCR induced errors found in product sequence      
Polymerase has low fidelity.Use a proofreading thermostable polymerase such as Platinum® Pfx DNA Polymerase. Note: The conditions for using Platinum® Pfx DNA Polymerase differ from other proofreading enzymes. Use a lower magnesium concentration (1 mM final), lower annealing temperature, and fewer units of enzyme (1.25 units). In addition, if products amplified with Platinum® Pfx DNA Polymerase remain in the well during electrophoresis, add SDS to the loading buffer to 0.1%. 
PCR induced errors found in product sequence
Too many cycles.
Reduce cycle number.
PCR induced errors found in product sequence 
The concentration of all four deoxynucleotides is not equal. 
Prepare a new deoxynucleotide mix and ensure that the concentration of all four nucleotides is equal, or use a prepared mix.
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References

  1.    Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus® 19, 46.
 
  2.    Chomczynski, P and Sacchi, N. (1987) Anal. Biochem. 162, 156.

  3.    Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 5294.
 
  4.    Gerard, G.F (1994). Focus® 16, 102.
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MAN0000941    11-Jun-2010