Application of oligo(dT) 30-latex for rapid purification of poly(A) + mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA

• Published in: Biochimica et biophysica acta Vol. 1156; no. 2; pp. 204 - 212
• Main Authors: Kuribayashi-Ohta, Keiko, Tamatsukuri, Shigeru, Hikata, Mikio, Miyamoto, Chikara, Furuichi, Yasuhiro
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 13.02.1993; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; DNA - biosynthesis; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; HeLa Cells; Humans; In Situ Hybridization; Latex; Nucleic acids; Oligo(dT)-latex bead; Oligodeoxyribonucleotides; Poly A - genetics; Poly A - isolation & purification; Poly dA-dT; Poly(A) + mRNA; Reverse transcriptase; RNA, Messenger - isolation & purification; RNA-Directed DNA Polymerase; Templates, Genetic; Vaccinia virus; Poly(A) + mRNA; Oligo(dT)-latex bead; Reverse transcriptase; Molecular hybridization; Purification; Messenger RNA; In situ; Analytical method; Oligonucleotide; Latex
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(93)90137-W

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 occured in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65°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.