In a PCR, these sequences are called primers and are short pieces of single-stranded DNA (approximately 15-30 bases). When designing a PCR experiment, the researcher determines the region of DNA to be ...

Ideally, primers should be 15-30 base pairs long (optimal for short amplicons, 70-200 bp). Design forward and reverse primers with similar melting temperatures (Tm), aiming for a Tm of around 60-65°C.

For example, a second primer pair that amplifies a “housekeeping” gene (i.e., a gene that has constant expression levels among the samples compared) can be included in the reaction (1, 2).

Melting temperatures (Tm) should be between 52-58°C. The difference in Tm for both forward and reverse primers should not be more than 5°C. The 3′ end of each primer should preferably be a G or C base ...

Variation in gene expression among cell types, tissues, and organisms is commonly examined by reverse transcription quantitative PCR, or RT-qPCR. In this process, RNA is isolated from samples of ...