title: Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.
abstract: The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.
authors.affiliation: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., Ibaragi, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kuribayashi-Ohta
authors.forename: S
authors.initials: S
authors.lastname: Tamatsukuri
authors.forename: M
authors.initials: M
authors.lastname: Hikata
authors.forename: C
authors.initials: C
authors.lastname: Miyamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Furuichi
mesh_terms: D004247:DNA / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D007840:Latex; D009838:Oligodeoxyribonucleotides; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification*; D011067:Poly dA-dT; D012333:RNA, Messenger / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase; D013698:Templates, Genetic; D014616:Vaccinia virus
chemical_list: D007840:Latex; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; C027903:oligo (dT); C013630:oligo (dT) poly (A); D011061:Poly A; D011067:Poly dA-dT; D004247:DNA; D012194:RNA-Directed DNA Polymerase