Purification of RT-PCR competent poly(A) mRNA from crude cell lysate by affinity precipitation.
Stimuli-responsive bioconjugates consisting of avidin covalently linked to poly(N-isopropylacrylamide) were used for the recovery of poly(A) mRNA hybridized to biotinylated poly(dT)-tags from crude cell lysates (Jurkat cells) by affinity precipitation. The bioconjugates are soluble in cold water but precipitate readily once a critical solution temperature (33 degrees C in pure water) is surpassed. The process is fully reversible and shows the expected dependencies on the composition of the aqueous solution and the bioconjugate chemistry. The results of the affinity precipitation were compared to those achieved with an accepted standard purification of poly(A) mRNA using avidin-activated magnetic beads. Both yield and quality/purity of the affinity precipitated poly(A) mRNA were found to be similar or better (especially removal of rRNA) than for poly(A) mRNA prepared by the magnetic particle-based protocol, while both mRNA isolates performed equally well in standard reverse transcriptase amplification (RT-PCR) of a beta actin transcript fragment. Poly(A) mRNA purification schemes based on affinity precipitation require no dedicated equipment and should have advantages in terms of scalability, handling, and costs.
Stocker
Gisela
G
Laboratory of Chemical Biotechnology, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland.
Vandevyver
Caroline
C
Hilbrig
Frank
F
Freitag
Ruth
R
D002474:Cell-Free System; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D005590:Fractional Precipitation; D012333:RNA, Messenger / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger