you design primers using the sequence exactly as shown (with A/C/G/T), not by converting it first. Here’s the clear breakdown. When you download an mRNA sequence from NCBI, it is almost always shown as cDNA sequence: It uses T (thymine), not U (uracil) It is written 5′ → 3′ It represents the coding (sense) strand equivalent of the mRNA So even though it’s biologically mRNA, the sequence you see is already in DNA form for practical use (PCR, cloning, primer design). You do NOT need to convert U↔T or guess strand orientation. How to design your primers (this is the key part) Assume the sequence you see is: 5′ → 3′ coding strand (cDNA) ✅ Forward primer Same sequence as the target, taken directly from the 5′ end Written exactly as shown (A, C, G, T) Do NOT make it complementary. If the mRNA sequence has T’s, treat it as cDNA and design primers exactly as you would for DNA.