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.
Ruiz
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Jules Stein Eye Institute, UCLA School of Medicine 90024.
Bok
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D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis; D018076:DNA, Complementary / Q000138:chemical synthesis; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D013698:Templates, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D008567:Membranes, Artificial; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D000254:Sodium-Potassium-Exchanging ATPase