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Purification of RT-PCR Competent Poly(A) mRNA from Crude Cell Lysate by Affinity Precipitation

Title data

Stocker, Gisela ; Vandevyver, Caroline ; Hilbrig, Frank ; Freitag, Ruth:
Purification of RT-PCR Competent Poly(A) mRNA from Crude Cell Lysate by Affinity Precipitation.
In: Biotechnology Progress. Vol. 22 (2006) Issue 6 . - pp. 1621-1629.
ISSN 1520-6033
DOI: https://doi.org/10.1021/bp060095u

Official URL: Volltext

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Engineering Science > Chair Process Biotechnology > Chair Process Biotechnology - Univ.-Prof. Dr. Ruth Freitag
Faculties
Faculties > Faculty of Engineering Science
Faculties > Faculty of Engineering Science > Chair Process Biotechnology
Result of work at the UBT: No
DDC Subjects: 500 Science
500 Science > 500 Natural sciences
600 Technology, medicine, applied sciences
600 Technology, medicine, applied sciences > 620 Engineering
Date Deposited: 25 Feb 2016 07:17
Last Modified: 25 Feb 2016 07:17
URI: https://eref.uni-bayreuth.de/id/eprint/31048