Application of oligo(dT)30-latex for rapid purification of poly(A)+ mRNA and for hybrid subtraction with the in situ reverse transcribed cDNA.
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.
Kuribayashi-Ohta
K
K
Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., Ibaragi, Japan.
Tamatsukuri
S
S
Hikata
M
M
Miyamoto
C
C
Furuichi
Y
Y
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
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