Materials and Reagents

BD 21 G needles (Fisher Scientific, catalog number: 14-823-55)
Manufacturer: BD, catalog number: 305274 .
BD tuberculin syringes (Fisher Scientific, catalog number: 14-823-2F)
Manufacturer: BD, catalog number: 309602 . This product has been discontinued.
TipOne 10 µl pipet tips (USA Scientific, catalog number: 1111-3200 )
TipOne 1-200 µl natural pipet tips (USA Scientific, catalog number: 1111-1800 )
TipOne 1,000 µl natural pipet tips (USA Scientific, catalog number: 1111-2020 )
Costar 6-well cell culture plates (Fisher Scientific, catalog number: 07-200-80)
Manufacturer: Corning, catalog number: 3506 .
1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 14-666-318 )
Iscove’s modification of DMEM (Mediatech, catalog number: 10-016-CV )
Tween 40 (CHEM-IMPEX INTERNATIONAL, catalog number: 01513 )
1x phosphate buffered saline (PBS) (MP Biomedicals, catalog number: 091860454 )
1x trypsin-EDTA (Mediatech, catalog number: 25-051-Cl )
0.5 M ethylenediaminetetraacetic acid (EDTA) pH 8.0 (Fisher Scientific, catalog number: BP2482-500 )
10% sodium dodecyl sulfate (SDS) (Mediatech, catalog number: 46-040-Cl )
Proteinase K (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: EO0491 )
Ribonuclease A, DNase free (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: EN0531 )
Potassium acetate (Fisher Scientific, catalog number: P171-500 )
Isopropanol (Fisher Scientific, catalog number: A416-500 )
Ethanol 70% (Fisher Scientific, catalog number: BP82011 )
Sodium hydroxide (NaOH) (Fisher Scientific, catalog: S318-500 )
SYBR Green PCR Master mix (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4309155 )
Magnesium chloride (MgCl₂) (Fisher Scientific, catalog number: BP214-500 )
Sucrose (Fisher Scientific, catalog number: S3-500 )
Tris-HCl buffer pH 7.4 (Lonza, catalog number: 51237 )
Potassium chloride (KCl) (Fisher Scientific, catalog number: P330-500 )
NP-40 10% solution (Thermo Fisher Scientific, catalog number: 28324 )
1 M MgCl₂ (see Recipes)
2.5 M sucrose (see Recipes)
TMK (see Recipes)
TMK + 1% NP-40 (see Recipes)
S1 buffer (see Recipes)
S2 buffer (see Recipes)
Cell lysis buffer (see Recipes)

Equipment

Ventilated microcentrifuge (Fisher Scientific, model: accuSpin^TM Micro 17R, catalog number: 13-100-676 )
Gilson PIPETMAN Classic pipets (Gilson, model: P1000, catalog number: F123602 )
Gilson PIPETMAN Classic pipets (Gilson, model: P20, catalog number: F123600 )
Gilson PIPETMAN Classic pipets (Gilson, model: P200, catalog number: F123601 )
Gilson PIPETMAN Classic pipets (Gilson, model: P2, catalog number: F144801 )
Water bath (Fisher Scientific, model: FS-14 )
Vortexer (Fisher Scientific, catalog number: 02-216-108 )
ABI Prism 7900HT sequence detection system (PE-Applied Biosystems) (Thermo Fisher Scientific, Applied Biosystems^TM, model: ABI PRISM^TM 7900HT )

Procedure

NP-40 fractionation
1. Wash plate 2 x with 2 ml PBS per well. Plate should have 5-10 x 10⁶ cells at 90-95% confluence.
2. Trypsinize cells (500 μl/well) and add 1 ml/well of media to neutralize. Combine the 6 wells of each plate. Can use a single 10 cm dish at the same confluence of 90-95% with 3-6 x10⁷ cells.
3. Spin cells at 4 °C, 100 x g for 5 min.
4. Resuspend cells with 1 ml PBS.
5. Pellet cells at 100 x g for 4 min at 4 °C.
6. Resuspend cells in 200 μl TMK.
  Note: For qPCR analysis of whole cell extraction, you can take out 50 μl and extract the DNA before proceeding to Procedure B.
7. Add 150 μl of TMK +1% NP-40.
8. Incubate on ice for 5 min with occasional gentle vortexing.
9. Pellet at 228 x g for 5 min at 4 °C.
10. Remove the supernatant without disturbing the pellet–This is the Cytoplasmic fraction.
11. Suspend the stripped nuclear pellet in 500 ml of S1 buffer.
12. Layer the S1 suspended pellets over 500 ml of S2 buffer.
13. Centrifuge at 1,430 x g for 5 min at 4 °C.
14. Aspirate supernatant off.
15. Resuspend the nuclei in 300 μl PBS.
  Note: At this point, all samples can be stored at 4 °C overnight before proceeding to Procedure B.
DNA extractions
1. Add 600 μl ice-cold cell lysis buffer to the cell extracts. Expect SDS precipitation with most extractions; it will not affect the end result. Vortex thoroughly for 30 sec.
2. Add 3 μl of Proteinase K solution (20 mg/ml). Incubate DNA sample at 55 °C for 3-16 h. The time is dependent on how much SDS precipitation is noticed on the samples and cell line used. A rule of thumb is the more SDS precipitation and larger cells tend to require more incubation time.
3. Shear DNA by drawing repeatedly (15 x) through a 21 G needle, avoiding excessive foaming and loss by aerosolization.
4. Allow cooling to RT, then add 3 μl of 4 mg/ml DNAse-free Ribonuclease A. Digest for 15-60 min at 37 °C.
  Note: At this point, all samples can be stored at 4 °C overnight before proceeding to step B5.
5. Cool to RT. Add 200 μl 5 M potassium acetate; vortex for 20 sec.
6. Pellet precipitated protein/SDS by centrifugation at 4 °C for 3 min at maximum RPM.
7. Transfer solution to a fresh tube with 600 μl isopropanol. Mix by inversion. Spin at maximum RPM for 3 min at RT.
8. Remove supernatant and wash DNA with 600 μl 70% EtOH. Spin for 3 min at maximum RPM to sediment the pellet. Aspirate the ethanol. Repeat with a 1-min spin and aspirate residual EtOH.
9. Air-dry for 15 min.
10. Redissolve the pellet in 100 μl 8 mM NaOH or 10 mM Tris. This may be slow–an incubation at 37 °C for 15 min is recommended.
11. The DNA may now be stored at -20 °C without incident.
Assessing efficiency of the NP-40 fractionation
To assess the accuracy of the nucleo-cytoplasmic separation, qPCR can be utilized using nuclear, mitochondrial and whole cell extract samples such as example below.
1. DNA samples were diluted 1 to 10 in dH₂O and incubated at 98 °C for 20 min followed by immediate cooling on ice.
2. Each PCR reaction contained 7.5 μl SYBR Green PCR Master mix (Fermentas), 2 μl of template, and 1 μM of each primer (Table 1) in a 15 μl reaction volume.
  
  Table 1. Primers for DNA amplification
3. QPCR was performed in triplicate for each sample using an ABI Prism 7900HT sequence detection system (PE-Applied Biosystems) for amplification and detection. PCR conditions were as follows:

Data analysis

The accuracy of the NP-40 nucleo-cytoplasmic fraction can be verified by comparing the amount of mitochondrial DNA copies detected in the nuclear fraction and the amount of β-globin DNA copies in the cytoplasmic fraction with the respective copies found in the total cell lysate. The data is analyzed by delta method which requires an average threshold cycle and standard deviation. Hence,
Ct = threshold cycle, a = nuclear or cytoplasmic sample, b = whole cell lysate sample:
The yield recovered is compared to cytoplasmic contamination in nuclear fractions. Figure 1 adapted from Galvis (2014) is an example of the data analysis graphs that can be composed with the obtained data. The ratio of mitochondrial DNA copies in the nuclear fractions to mitochondrial DNA copies in the whole cell lysate. The example demonstrates a successful fractionation. The nuclear fraction has a 1:1 ratio of nuclear DNA with the whole cell extract; while at the same time there is minimal mitochondrial DNA in comparison to the whole cell extract. The cytoplasmic fraction contains minimal nuclear DNA in comparison to the whole cell extract while it contains an almost 1:1 ratio of mitochondrial DNA with the whole cell extract.
Note that the recommended inclusion data is that all triplicates must be within one cycle apart from each other and all obtained cycles must be smaller than the negative control that need to be included in the qPCR runs.

Figure 1. Sample data analysis of fractionation protocol. This is a ratio of mitochondrial and β-globin DNA in nuclear and cytoplasmic fractions vs. total cell lysates. GHOST-R5X4 cells were transfected either with DBR1 shRNA pHyper-D4 (D4) or DBR1 shRNA triple mismatch, pHyper-M4 (M4). Two to twenty-four hours later, the cells were either fractionated into nuclear and cytoplasmic fractions or lysed to prepare whole cell extracts. DNA was isolated for qPCR to evaluate the success of fractionation. A. β-Globin primers were utilized to assay the exclusion of nuclear contamination in the cytoplasmic fraction in contrast to whole cell extracts. The graph illustrates the ratio of β-globin copies in the cytoplasmic fractions to β-globin copies in the whole cell lysates. B. β-Globin primers were utilized to assay concentration of nuclear fractions compared to whole cell extracts. The graph illustrates the ratio of β-globin copies in the nuclear fractions to β-globin copies in the whole cell lysate. C. Mitochondrial primers were used to assay concentration of cytoplasmic fraction compared to whole cell extracts. The graph illustrates the ratio of mitochondrial DNA copies in the nuclear fractions to β-globin copies in the whole cell lysates. D. Mitochondrial primers were utilized to assay the exclusion of mitochondrial contamination in the nuclear extractions.

Notes

The data presented in Procedure C were the unpublished controls for experiments denoted in Galvis et al., 2014. Debranching enzyme 1 (DBR1) in our experiments has been implicated in reverse transcription of the HIV-1 genome.
In Procedure A step 12, it is very important not to disturb the pellet and hence it is better to leave some of the supernatant behind. The purpose of the subsequent steps is to remove the remaining mitochondrial DNA.
In our experience, the resuspension of the DNA with 8 mM NaOH has given us better overall recovery yield for downstream applications in comparison to 10 mM Tris. However, if the experiments desired only need for qPCR purposes only and not sequencing then 10 mM Tris is sufficient for resuspension of the final pellet.

Recipes

1 M MgCl₂ (500 ml)
Add 101.65 g MgCl₂·6H₂O into 500 ml ddH₂O
Store at room temperature
2.5 M sucrose
8.55 g of sucrose
Up to 10 ml with ddH₂O
Need to heat to 37 °C in order to have sucrose fully dissolve
Store at room temperature
TMK (10 ml; store in 4 °C)
TMK + 1% NP-40 (2 ml)
1.8 ml TMK
200 μl 10% NP-40
Store in 4 °C
S1 buffer
0.25 M sucrose
10 mM MgCl₂
S2 buffer
0.35 M sucrose
0.5 mM MgCl₂
Store at 4 °C
Cell lysis buffer
10 mM Tris-HCl, pH 7.4
0.1% SDS
1 mM EDTA
Store at 4 °C

Acknowledgments

This work was supported in part by grant ID07-I-124 from the California HIV-AIDS Research Program to David Camerini. Support of the UCI Center for Virus Research, UCI Cancer Research Institute and the Chao Family Comprehensive Cancer Center are acknowledged. Alvaro Galvis was supported by the UCI Medical Scientist Training Program, grant T32-GM08620. This protocol is adapted and modified from the original in Sambrook and Russell, 2001.

References

Galvis, A. E. (2014). An RNA lariat intermediate in HIV-1 cDNA synthesis. ProQuest 3615193.
Galvis, A. E., Fisher, H. E., Nitta, T., Fan, H. and Camerini, D. (2014). Impairment of HIV-1 cDNA synthesis by DBR1 knockdown. J Virol 88(12): 7054-7069.
Sambrook, J. and Russell, D. W. (2001). Molecular cloning: a laboratory manual. CSHL Press.