RNA-SIP fraction precipitation
- GlycoBlue™ Coprecipitant (15 mg ml-1); note: non-RNA-grade glycogens may contain residual RNA). Other RNA-grade glycogens may also be used but the pellet will most likely not be visible.
- 100% EtOH (molecular grade)
- 7. 5 M NH4-Acetate (RNase free)
- 70% EtOH (molecular grade in RNase free)
- Optional: RNA Storage Solution (Ambion)
cDNA synthesis (only RNA-SIP)
Methods
Gradient preparation
- Prepare all solutions in advance.
- Equilibrate the CsTFA solution to room temperature for about 60 min.
- Calibrate the refractometer with pure water.
- For each sample, make the following mixture in a 50 ml tube: 4.04 ml CsTFA 795 µl GB (adjust volumes according to the ultracentrifugaion-tube volume).
- Mix by inverting and measure the density on a refractometer (typically using 40--50 µl). The density should be1.3702 ± 0.0002 nD-TC.
- Add 3.59 % vol. HiDi.
- Measure the density again. The density should be: 1.3725 ± 0.0002 nD-TC
- Transfer apporx. 4.8 ml of the mixture to each centrifugation tube using a micropipette. Make sure the volume reaches only the bottom of the neck.
- Add the RNA. Typically, 300--500 ng RNA are enough to recover enough material for PCR after centrifugation and fractionation. Do not exceed ca. 1250 ng (ca. 250 ng per 1 ml of total volume), since this might casue the RNA to precipitate and distort the gradient.
- Balance tube pairs to within 0.1 g, together with caps and spacers.
- Close the caps.
- Place tubes in the rotor and close only the ones with the tubes using the torque wrench up to about 13.6 N m (120 in.-lb).
- Centrifuge at 130,000 g (42,400 rpm for the VTi 90 rotor) at 20 °C for 65 h at maximum accelaration and minimum deceleration.
Gradient fractionation
- Fill a 20 ml syringe with RNase-free water; remove air bubbles.
- Attach the tube to the syringe and mount it on the pump.
- Set volume to 0.75 ml min-1 and collect in 20 s steps, this will yield 20 fractions (alternatively, set volume to 0.45 ml min-1 and collect in 40 s steps for fewer fractions). Volume – off and diameter – 2.2 cm.
- Connect a new 23G needle to the tube and test flow.
- Stop the ultracentrifuge.
- Prepare 20 1.5 ml non-stick tubes per gradient in a rack.
- Carefully carry the centrifuge rotor to the lab with minimum disturbance, mount 1 ultracentrifugation tube on the utility clamp about 1 cm above the opening of the collection tubes.
- Carefully insert the needle connected to the pump’s tube horizontally. Make sure no air bubble is trapped in the needle or the tube. Press the needle slightly against the opposing tube wall to fix it in its place without puncturing a hole in that side of the tube.
- Using a new 26G needle, carefully puncture a hole at the bottom of the ultracentrifugation tube and remove the needle. The ultracentrifugation tube should not leak at this stage.
- Place the rack under the ultracentrifugation tube so that it drops to the first collection tube.
- Start the pump, observe the first drop falling and immediately start the stopwatch.
- After 20 s, shift the rack so that the solution drops to the second collection tube. Continue in a similar fashion until all tubes are filled.
- After finishing fractionating all ultracentrifugation tubes, measure the density of every fraction by pipetting 40 µl to the refractometer. The density of the fractions should decrease at a constant rate as you progress from the lighter to the heavier fraction.
- To each tube add 2 µl of glycogen, 2.5 volumes of 100% EtOH and 0.1 volumes 5M NH4-acetate (assuming 250 µl fractions were collected, add 21 µl 5M NH4-acetate and 525 µl 100% EtOH). Incubate for 30 min at -80 °C.
- Centrifuge at 14,000 rpm for 30 min at 4 oC.
- Decant the supernatant, wash once with 1 ml of ice cold 75% EtOH, invert the tube several times. Centrifuge at 14,000 rpm for 15 min.
- Remove as much as possible from the supernatant first using a 1 ml tip, spin down the remaining drops in the tube, and remove them with a 100 µl tip. Once again, be careful not to pipette the pellet with the liquid.
- Leave tubes open at room temperature for no more than 5 min (preferably under flame or a biological hood) in order to evaporate the remaining ethanol (note: pellets might not be completely dry at this point). Alternatively, pellets can be dried under a filtered stream of N2.
- Resuspend the pellets in 10 µl RNase free water or the RNA Storage Solution.