Direct RT-PCR amplification of mRNA supported on membranes

We describe a simple and efficient technique that facilitates the amplification of specific mRNA for cloning and sequencing purposes. An mRNA bound to a small piece of membrane filter is used as a template to synthesize complementary DNA. The product of this reaction is then transferred to a new tube and amplified using a standard PCR protocol. By simple enzymatic treatment, this RNA membrane can be reused as many times as needed with no problems of low yield, mispriming or background. Multiple advantages and different applications can be gained with this procedure. We have been using this technique to characterize a 4.5-kb mRNA from human retinal pigment epithelial cells following identification by Northern blot. According to the size of the PCR amplification products, this mRNA band contains portions of the coding sequence for the Na+K(+)-ATPase beta 1 subunit.