How should I design the miRNA qRT-PCR primer to be used with the NCode™ system?
Invitrogen has already designed primer sequences that you may use for each miRNA on our arrays. These can be found within the NCode™ database. The Tm of the primers was calculated using nearest-neighbor approximation based on the following reference: John SantaLucia, Jr. Hatim T. Allawi, and P. Ananda Seneviratne. Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability. Biochemistry 1996, 35:3555-62. If you are designing a primer for a unique or novel small RNA, please consider the information below: The optimal Tm range is 58–64 °C. For sequences with calculated Tm values less than 58 °C, either “A”s may be added to increase Tm, or modified bases could be substituted. No more than 6 “A”s should be added to any one sequence. If an increase in Tm is still needed, GC residues can be added to the 5’ end. The maximum primer length designed was 30 nucleotides. For sequences with calculated Tm greater than 64 °C, bases were truncated from the 5’ end and the resulting primer
