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  • Quantitation of Isolated Poly(A)+ RNA: From the Magnetic mRNA Isolation Kit

    Overview

    Isolation Preparation

    Allow all kit components to come to room temperature.

    Resuspend Oligo d(T)25 beads by agitating at room temperature (RT) for 30 minutes.

    Reuse Oligo d(T)25 beads for second-round poly(A)+ selection.

    Oligo d(T)25 beads can be re-used for a second-round of purification of a poly(A)+ RNA eluent without regeneration. Wash beads once with an additional 100 μl of elution buffer. Place in magnetic rack and pull beads to the side of the tube. Remove and discard wash solution. Wash beads once with 200 μl of Lysis/Binding Buffer then re-apply previously isolated eluent to beads after adjusting salt concentration to 0.5 M LiCl or NaCl. Repeat isolation steps.

    Introduction

    Regeneration of Oligo d(T)25 beads.

    Oligo d(T)25 beads can be regenerated and used to isolate RNA from a different source. Add 0.1 NaOH to the beads and incubate at room temperature with agitation for 5 minutes. Wash beads twice with sterile RNase-free 1X PBS (pH 7.4) containing 0.1% Tween 20. Then store in the same buffer.

    Quantification of poly(A)+ RNA can be done by measurement of the A260 absorbance using a NanodropTM spectrophotometer to avoid dilution of the sample.  The A260/280 ratio should be 1.6 or greater.

    The amount of isolated RNA will vary with the source of the RNA sample, typically for RNA 1 A260 Unit = ~40 μg/ml.

    The eluted mRNA should be placed in the magnetic rack while removing aliquots for quantification to avoid pipetting of beads, which will interfere with spectrophotometric analysis.