Optimal Primer design for RT-PCR


Choosing primers for reverse transcription

The primer used for reverse transcription affects both the size and the specificity of the cDNA produced. Four kinds of primers are commonly used in RT-PCR, each with specific advantages.

Type of
RT Primer
Hybridizes to: Advantages/Comments
Oligo(dT)N Endogenous poly(A) tail found at the 3' end of eukaryotic (e.g., mammalian) mRNA.

Generates full-length cDNA

If template contains oligo(A) stretches internally, the primer may bind these, leading to mispriming and transcripts that are not full-length.

Prevent transcription of large poly (A) stretches.

Anchored Oligo(dT)N Very beginning of the poly(A) tail.

Generates full-length cDNA.

Preferred priming method for most two-step RT-PCR applications.

Part of the Transcriptor First Strand cDNA Synthesis Kit

Random Hexamer Many sites throughout the length of an RNA (mRNA, tRNA or rRNA).  

Provides uniform representation of all RNA sequences in mRNA.

Can prime cDNA transcription from RNAs that do not contain a poly(A) tail.

Short cDNA transcripts may be the ideal way to overcome difficulties presented by RNA secondary structures.

Adjusting the ratio of random primers to RNA in the RT reaction controls the average length of cDNAs formed.

Note: A high ratio of random hexamers and RNA template will increase the chances of amplifying the entire target sequence.  

Sequence-specific Only sequences exactly complementary to the primer sequence

Selects for a particular RNA.

Greatly increases the specificity and sensitivity of the RT-PCR.

This is the only type of priming that can be used for one-step applications.

Tip: Random primers should be used at a final concentration of 60 µM for an optimal reaction result.

If the fragment of interest is located on the 5’ end, then a mix of random hexamers and anchored oligo(dT)N can give the optimal result. Typically, 60 μM of random hexamers and 2.5 μM of anchored oligo(dT)N are used.

Designing mRNA specific Primers

RT-PCR amplification of a particular RNA sequence requires two PCR primers that are specific for the gene transcript of interest. The primer design should allow differentiation between the amplified product from cDNA and an amplified product derived from contaminating genomic DNA.

There are two approaches to designing the required primers (Figure 1):

Panel 1. Make primers that anneal to sequences in exons on both sides of an intron (Figure 1, panel 1). With such primers, any product amplified from genomic DNA will be much larger than a product amplified from intron-less cDNA.
Panel 2. Make primers that span exon/exon boundaries on the RNA (Figure 1, panel 2). Such primers span more then one intron and therefore should not amplify genomic DNA.

Primer design for RT-PCR 
Figure 1: Two approaches to primer design. For explanation of panels, see text.


Choosing RT-PCR enzymes

Obviously, a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. Among the enzyme properties to consider are:

  • Maximum length of template that can be transcribed into full-length cDNA
    Some reverse transcriptases can only transcribe short templates (less than 3 kb). Others can transcribe much longer templates (up to 14 kb). Be sure to select an enzyme that is likely to produce a full-length cDNA from your chosen RNA target.
  • Temperature optimum
    Higher incubation temperatures help eliminate problems of template secondary structure and decrease false priming from sequence-specific primers. Thus, reverse transcriptases that can be incubated at high temperatures are more likely to produce accurate copies of the RNA target, especially if it has a high GC content.
  • Sensitivity
    Reverse transcriptases differ in their ability to copy small amounts of template. This is especially important when the chosen target is expressed at a very low level.
  • Specificity
    Reverse transcriptases also differ in their ability to prepare accurate, full-length cDNA copies from difficult templates (e.g., those with large amounts of secondary structure).
  • Incorporation of modified nucleotides
    If the cDNA is to be used in microarray analysis, the RTase must be able to incorporate several modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, DIG-, and biotin-dUTP).
  • RNase H activity
    RNase H removes the RNA from the RNA:cDNA hybrid. This can increase the sensitivity of the subsequent PCR. However, unless carefully controlled, RNase H activity may compete with cDNA synthesis, resulting in loss of template before full-length cDNA is transcribed. RNases have differing amounts of RNase H activity; some enzymes lack this activity.


Optimizing the reverse transcription procedure

If you are working with difficult RNA templates with secondary structures, such as GC-rich templates, you can successfully reverse transcribe them using the following procedures:

One-step RT-PCR:

  • Briefly denature the RNA template at +94°C (1 minute) before adding it to the reaction mixture.
  • Increase the denaturation temperature, or the denaturation time in each of the PCR cycles.
  • If using DMSO in your reaction mix, an optimization is required (up to 10%).

Tip: Do not incubate the reverse transcriptase enzyme or RNase inhibitor at this elevated (+94°C) temperature,even briefly, since both will be inactivated.

Two-step RT-PCR:

  • Denature the template-primer mixture for 10 minutes at +65°C before adding reverse transcriptase.
  • Use random hexamer primers or gene-specific primers.
  • Use reverse transcriptases which allow reverse transcription at higher temperatures, such as the Transcriptor First Strand cDNA Synthesis Kit.



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