1. When the total RNA is reverse-transcribed, the cDNA corresponding to the mRNA has one strand identical to the mRNA sequence, and the other strand (rc) is a template strand. It should be clear when you draw it on paper, with 20 bases starting from ATG in mRNA sequence (intentional chain) as forward primers and 20 bases starting from template chain TCA (reverse complementary sequence of TGA) as reverse primers.

2. Because the amplification of coding sequence requires strict primer positions (from ATG to TGA), the designed primers usually have low scores (primer dimer has been formed or there is a serious mismatch), which sometimes affects the amplification of the target product. I sometimes design a pair of primers first, which are in the 5' and 3'UTR regions respectively, and design them with software to ensure that the primers are easy to use; In this way, cDNA is used as a template to amplify, and then this product is used as a template to amplify with a pair of primers from ATG to TGA, which largely avoids mismatch and is easier to amplify successfully.