Design Guidelines
Tm prediction
Two models for Tm prediction
QIAGEN uses two different models for predicting the melting temperature (Tm) of LNA oligonucleotides:
- DNA Tm: This model predicts the Tm of a complex formed between the LNA oligo and a complementary DNA strand.
- RNA Tm: This is a new model that predicts the Tm of a complex formed between an LNA oligo and a complementary RNA strand.
Both models are based on experimental data from thousands of oligonucleotide hybridizations. For oligonucleotides of 15–27 nucleotides in length, the resulting predictions have a precision of 1.70 and 2.07°C for RNA and DNA targets, respectively.
Recommended use of the models
We recommend using the RNA Tm for applications in which LNA oligos target RNA sequences. RNA Tm information will be introduced to relevant products in the future.
We generally recommend starting with a hybridization temperature 30°C below the RNA Tm when detecting RNA targets. This temperature translates to approximately 20°C below the DNA Tm. For example, the oligo C+TG+AC+CGT+ATG+GTC+TA+TA will have RNA Tm and DNA Tm of 86 and 70°C, respectively.
Please note that the actual hybridization temperatures may have to be optimized for each experiment.
We generally recommend starting with a hybridization temperature 30°C below the RNA Tm when detecting RNA targets. This temperature translates to approximately 20°C below the DNA Tm. For example, the oligo C+TG+AC+CGT+ATG+GTC+TA+TA will have RNA Tm and DNA Tm of 86 and 70°C, respectively.
Please note that the actual hybridization temperatures may have to be optimized for each experiment.
Assumptions and limitations of the models
- The Tm calculation models are based on a modified nearest-neighbor thermodynamic model combination and data from measurements of oligonucleotides ranging from 15–27 nucleotides in length.
- All predictions are based on the following:
- Oligos range from 6–80 bases in length
- Salt concentration (Na+) is 115 mM
- Solution is neutral (pH 7–8)
- Oligo concentration is 1 and 2 µM for RNA and DNA Tm calculations, respectively.
- The calculations do not account for effects of divalent cations, triphosphates or chemical modifications except LNA
- 2’ O-Me modifications can be expected to increase the Tm by 1.3°C per inserted modification. The influence of such modifications has not been taken into account in the models.