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Chapter 2 | rRNA Reduction and Preparation of Total RNA with Diluted Poly-A RNA Controls
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Procedure B: Preparation of Hybridization Buffer with Betaine
This Procedure requires the use of the RiboMinus Human/Mouse Transcriptome
Isolation Kit that needs to be obtained directly from Invitrogen.
IMPORTANT: The protocol for using the RiboMinus Kit for rRNA reduction for
GeneChip® arrays has been modified from the manufacturer’s original
recommendation. Follow only the procedure described in this manual for
optimal transition to the target labeling process immediately following the
rRNA reduction steps.
1. Prepare the buffer by mixing these components as listed in Table 2.2. Keep tube at
room temperature.
Table 2.2 Hybridization Buffer with Betaine
Starting Total RNA Amount
*
1 μg
2 μg
Component
Volume for 1 Rxn
Volume for 1 Rxn
Betaine, 5M
54 μL
84 μL
Invitrogen Hybridization buffer
126 μL
196 μL
Total Volume
180 μL*
280 μL*
A 30 μL overfill is included in the Total Volume, sufficient for completing the RiboMinus procedure for a single total RNA sample.