OP-HELISCOPE-CAGE-v3.12

Protocol: OP-HELISCOPE-CAGE-v3.12

Author: Kojima, Miki

Created: Nov. 25, 2010

Update:

Parameters:

Description:

Sample preparation unit is 8 strips.

Reagent and sample volume are showing the amount per 1 well. Therefore when preparing a sample, the volume is calculated at 8-fold.

Step1, First-strand cDNA synthesis:

First-strand cDNA was synthesized using total RNA, whose RIN number of Bioanalyzer and RNA 6000 Pico kit (Agilent) is greater than 7, and whose concentration is more 0.5 µg/µl. Each aliquot of 5 µg of total RNA was mixed with 100 pmol of random N15 primer and added RNase free water up to 6 µl. The RT enzyme mix contained 1x SuperScript III reaction buffer, 10 nmol dNTP, 0.65 M sorbitol, 0.14 M trehalose solution, 0.05 mM DTT, 750 units of SuperScript III (Invitrogen) for a total volume of 32 µl for one reaction and was kept on ice until use. The RNA/primer mix was denatured at 65°C for 5 min, then immediately chilled on ice. After all liquid was collected by centrifugation, 32 µl of RT enzyme mix was added and the solution was gently mixed on ice. The reaction mixture was incubated at 25°C for 30 sec, 42°C for 30 min, 50°C for 10 min, 56°C for 10 min, and 60°C for 10 min, then chilled at 4°C. The cDNA/RNA hybrids were purified by AMPure RNA Clean XP beads (Beckman Courlter) [1]. The sample was eluted to 40µl.

Step2, Oxidation and biotinylation:

40 µl of purified cDNA/RNA hybrid was mixed with 2 µl of 1 M sodium acetate (pH 4.5) and 2 µl of freshly prepared 250 mM sodium periodate then chilled on ice for exactly 45 min in the dark. The reaction was stopped by addition of 2 µl of 40% glycerol and 14µl of 1M Tris HCl pH 8.5. Sample was purified by AMPure RNA Clean XP beads [1], eluted to 40µl. 1 µl of 1 M sodium acetate (pH 6.0) and 4 µl of freshly dissolved 10 mM biotin (long arm) hydrazide (VECTOR Lab.) were added to sample solution. The mixture was incubated for 2 h at 23°C in the Thermal cycler. The reaction solution was added 12 µl of Isopropanol and purified by AMPure RNA Clean XP beads [1], eluted to 40µl. 4.5 µl of 10x RNase I buffer and 0.5 µl of 10 unit/µl RNase I (Promega) were added and the solution incubated at 37°C for 30 min.

Step3, Capture and release:

50 µl of MPG streptavidin magnetic beads solution (TAKARA Bio) was mixed with 0.625 µl of 20 µg/µl tRNA (Sigma), then stand on ice for 30 min with occasional mixing to block the bead surfaces. The beads were separated using a magnetic tube stand, then twice washed with 100 µl of wash buffer 1 containing 4.5 M sodium chloride and 50 mM EDTA (pH 8.0), followed by suspension in 105 µl of the same buffer with 12.5 µg of tRNA included. All 105 µl of magnetic beads was mixed with 45 µl of cDNA/RNA hybrid sample and incubated at 37°C for 30 min with mixing every 2 min. The beads were separated from supernatant using a magnetic stand, then washed with 150 µl of the following buffers: once with wash buffer 1, once with wash buffer 2 containing 0.3 M sodium chloride and 1 mM EDTA (pH 8.0), twice with wash buffer 3 containing 1 mM EDTA, 0.4% sodium dodecylsulfate, 0.5 M sodium acetate, and 20 mM Tris-HCl (pH 8.5), and twice with wash buffer 4 containing 1 mM EDTA, 0.5 mM sodium acetate, and 10 mM Tris-HCl (pH 8.5). The captured cDNA/RNA was suspended in 35 µl of 1x RNase I buffer and incubated for at 95°C for 5 min, then on ice immediately. The beads were separated using a magnetic stand and the supernatant is transferred to new tube. 35 µl of 1x RNase I buffer was added to released beads again for wash. The beads were separated using a magnetic stand and the supernatant is transferred to new tube again. Total volume of released cDNA is 65 µl, then 3 µl of 2 unit/µl RNase H (Invitrogen) and 2 µl of 10 unit/µl RNase I (Promega) were added and the solution incubated at 37°C for 15 min. The reaction solution was purified by AMPure XP beads [1], eluted to 40 µl. 2 µl of aliquot was used for the quality control by measuring concentration (Oligreen).

Step4, RNase I: cDNA sample was 40 µl, then 4.5 µl of 10x RNase I buffer and 0.5 µl of 10 unit/µl RNase I (Promega) were added and the solution incubated at 37°C for 30 min. The reaction solution was purified by AMPure XP beads [1], eluted to 40 µl.

Step5, Sample concentration:

40 µl of cDNA sample was concentrated to 12 µl by Speed Vac. 1 µl of aliquot was used for the quality control by measuring concentration (Oligreen) and qPCR respectively.

Reference/Notes:

[1] The AMPure beads which 1.8-fold sample volume is added to sample solution. Mix by pipetting 10 times and incubate for 10 min at room temperature. Sample mix and incubation is repeated more 2 times. The beads were separated from supernatant using a magnetic stand, and washed with 200 µl of 70% Ethanol twice. 40 µl of sterile water is added and pipetting 60 times for resolve the beads. Solved beads solution was incubated at 37°C for 5 min. The beads were separated from supernatant using a magnetic stand, transfer the supernatant to new tube as purified sample solution. Purified sample solution was incubated at 37°C for 10 min for remove the contaminated Ethanol.