DIRECT RT-PCR AMPLIFICATION OF MESSENGER-RNA SUPPORTED ON MEMBRANES

We describe a simple and efficient technique that facilitates the ampl ification of specific mRNA for cloning and sequencing purposes. An mRN A bound to a small piece of membrane filter is used as a template to s ynthesize complementary DNA. The product of this reaction is then tran sferred to a new tube and amplified using a standard PCR protocol. By simple enzymatic treatment, this RNA membrane can be reused as many ti mes 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 identifica tion 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 beta1 subunit.