UNIT 4.10 Identification of Newly Transcribed RNA


Quality of nuclei used in nuclear runoff protocols is a major determinant in the success of the experiment. Normal lymphocytes in particular can present a problem because the nuclei are more fragile. In this protocol, cells are resuspended in an isoosmotic buffer containing nonionic detergent, then lysed by Dounce homogenization. Nuclei are collected by ultracentrifugation through a sucrose cushion and are quite clean and free of contaminating membranes and cytoplasmic components. Typically, 10% to 30% more [a-32P]UTP is incorporated into nascent transcripts of nuclei prepared by this method. The density of nuclei varies with cell type so a pilot experiment should be performed to verify that these conditions (which work well for murine splenic lymphocytes and other vertebrate cells) result in a nuclear pellet.

Additional Materials

For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see Sucrose buffer I (see recipe), ice cold
Sucrose buffer II (see recipe)

Dounce homogenizer with B pestle, ice cold
Polyallomer centrifuge tubes ( 9/16 ´ 3 3/4 in., Beckman) for SW 40.1 rotor
Ultracentrifuge and SW 40.1 rotor or equivalent
1.5-ml microcentrifuge tubes, chilled on dry ice

NOTE: Keep cells and nuclei on ice until the nuclei are frozen.

Harvest and lyse cells

1. Harvest and wash cells as in basic protocol step 1.

2. Loosen cell pellet by gently vortexing 5 sec. Resuspend cell pellet in 4 ml ice-cold sucrose buffer I. Examine a small aliquot of cells for lysis with a phase-contrast microscope.

Many cell types will lyse at this point and do not require Dounce homogenization. If cells have lysed, proceed directly to step 4.

3. Transfer cells to an ice-cold Dounce homogenizer and break the cells with five to ten strokes of a B pestle or until the nuclei appear free of cytoplasmic tags. Check a few microliters of cells with a phase-contrast microscope to be sure they are uniformly lysed.

4. Transfer nuclei to a clean 50-ml conical polypropylene centrifuge tube and add 4 ml sucrose buffer II. Mix by gentle pipetting and inversion.

The final concentration of sucrose in cell homogenate should be sufficient to prevent a large buildup of debris at the interface between homogenate and sucrose cushion. The amount of sucrose buffer II added to cell homogenate may need to be adjusted.

Collect nuclei

5. Add 4.4 ml sucrose buffer II to polyallomer SW 40.1 tube.

Sucrose buffer II serves as the sucrose cushion. Unlysed cells will not sediment through the sucrose cushion. If these conditions do not result in a nuclear pellet, adjust the concentration of sucrose in sucrose buffer II.

6. Carefully layer nuclei (from step 4) onto the sucrose cushion. Use sucrose buffer I to top off the gradient.

Do not centrifuge more than 2 ´ 108 nuclei per tube.

7. Centrifuge the gradient 45 min at 30,000 ´ g (15,500 rpm in SW 40.1 rotor), 4°C.

8. Remove supernatant by vacuum aspiration. Tilt the tube sideways so supernatant is pulled away from the pellet and remove any bubbles or liquid that remain on the side of the tube. Return tube to an ice bucket.

Nuclei should form a tight pellet at the bottom of the tube and there may be some debris caught at the interface between sucrose buffers I and II. If the cells did not lyse during Dounce homogenization, nuclei will not pellet. Thus, it is important to be sure that the majority of the cells are clearly lysed in step 3. If the pellet appears as a gelatinous mass, nuclei have lysed and the pellet should be discarded.

9. Loosen nuclear pellet by gently vortexing 5 sec. Add 200 ul ice-cold glycerol storage buffer per 5 ´ 107 nuclei and resuspend nuclei by pipetting up and down.

Nuclei will be clumped at first but will disperse with continued pipetting. Pipetting should be steady but should not create air bubbles.

10. Aliquot 210 ul (~5 ´ 107 nuclei) into chilled microcentrifuge tube and immediately return tube to dry ice. Store frozen nuclei at -70°C or in liquid nitrogen.

Frozen nuclei are stable for at least 1 year.

11. Proceed with nuclear runoff transcription assay starting at basic protocol step 5.

From Current Protocols in Molecular Biology Online
Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.