Materials and reagents

Biological material

GM12878 cell line

Reagents

Dulbecco’s phosphate-buffered saline (PBS) (Fisher Scientific, catalog number: BP399-1)
Trypsin-EDTA solution (0.5%) (Gibco, catalog number: 15400054)
Tissue culture media: Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, catalog number: 11875093)
Fetal bovine serum (FBS) (Gibco, catalog number: 10270106)
Penicillin-Streptomycin (Gibco, catalog number: 15140122)
Trypan blue (Alfa Aesar, catalog number: A1860)
Tris-Base (Fisher Scientific, catalog number: BP152-5)
Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-1)
Magnesium chloride (MgCl₂) (Fisher Scientific, catalog number: BP214-500)
DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)
NP-40 (Sigma-Aldrich, catalog number: 9016-45-9)
Ribonucleoside vanadyl complexes (NEB, catalog number: S1402S)
Sodium acetate (Fisher Scientific, catalog number: BP333-500)
Molecular grade ethanol (Merck, catalog number: 64-17-5)
TRIzol (Life Technologies, catalog number: 15-596-018)
Chloroform:isoamyl alcohol (1:24) (Fisher Scientific, catalog number: BP1752I-400)
Molecular grade isopropanol (Fisher Scientific, catalog number: BP2618-1)
RNase-free water (Macherey Nigel, catalog number: 740378)
10× M-MuLV RT buffer (NEB, catalog number: #M0253)
DNase I (NEB, catalog number: #M0303)
M-MuLV reverse transcriptase (NEB, catalog number: #M0253)
RNase inhibitor (NEB, catalog number: #M0314)
dNTP mix (Thermo Scientific, catalog number: R0192)
Random hexamer (Invitrogen, catalog number: N8080127)
Primers (IDT, origin: USA)
SYBR Fast qPCR Master Mix (KAPA Biosystem, catalog number: KK4602)

Solutions

Complete media for GM12878 cell line (see Recipes)
Hypotonic buffer (see Recipes)
RNA precipitation solution (RPS) (see Recipes)

Recipes

Complete media for GM12878 cell line
Reagent Stock concentration Final concentration Volume (mL)
RPMI media 395
FBS 100% 20% 100
Penicillin-Streptomycin 10,000 U/mL 100 U/mL 5
Total 500

Hypotonic buffer

Reagent	Stock concentration	Final concentration	Volume (μL)
Tris (pH 7.5)	100 mM	10 mM	500
KCl	100 mM	10 mM	500
MgCl₂	150 mM	1.5 mM	50
DTT	100 mM	0.5 mM	25
NP-40	10%	0.075%	37.5
Ribonucleoside vanadyl complexes	200 mM	2 mM	50
H₂O			3837.5
Total			5,000

RNA precipitation solution (RPS)
Reagent Stock concentration Final concentration Volume (mL)
Sodium acetate (pH 5.5) 3 M 150 mM 0.5
Ethanol 99.5% 9.5
Total 10
Final concentration of each component is 0.15 M sodium acetate (pH 5.5) in ethanol.
Keep at -20 °C for storage.

Equipment

Refrigerated tabletop centrifuge (15 or 50 mL conical tube adaptors) (Thermo Scientific, model: Multifuge X1R)
Refrigerated benchtop centrifuge (1.5 mL tube rotor) (Eppendorf, model: 5424R)
Light microscope (Olympus, origin: USA)
Vortexer (Biosan, model: V-1 plus)
Micropipettors (Gilson, origin: USA)
NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific, origin: USA)
Applied Biosystems Veriti Dx 96-Well Thermal Cycler PCR Thermocycler (Applied Biosystem, origin: USA)
Bio-Rad Connect Real-Time PCR System (Bio-Rad, origin: USA)

Procedure

Subcellular fractionation
1. Cell harvesting from culture plates
  1. Grow the cells to a confluency of ~80%. Rinse the cells with 1× PBS.
  2. For adherent cells, detach the cells with Trypsin-EDTA. For suspension cells, proceed to step A1d.
  3. Add an equal volume of complete media to stop the trypsin proteolysis.
  4. Collect the cell suspension in a conical tube and centrifuge at 200× g for 5 min.
  5. Remove the supernatant and resuspend the cell pellet with 1× PBS.
  6. Perform cell counting and aliquot 1 × 10⁶ cells into a 1.5 mL microcentrifuge tube.
  7. Centrifuge the tube at 200× g for 5 min and remove the supernatant.
  8. Keep the cell pellet on ice prior to the next step.
2. RNA isolation from cytoplasmic and nuclear fractions of cells
  1. Resuspend the cell pellet gently with 100 μL of ice-cold hypotonic buffer and incubate on ice for 5 min.
    Note: Volume of the hypotonic buffer can be adjusted based on the size of cell pellet.
  2. Check for membrane lysis with trypan blue. Proceed with centrifugation at 500× g for 10 min at 4 °C until > 90% of cells are lysed. Otherwise, increase the incubation time for step A2a until > 90% of cells are lysed.
    Note: Do not overlyse the cells.
  3. Collect the supernatant (cytoplasmic fraction) and wash the pellet (nuclear fraction) with 300 μL of ice-cold hypotonic buffer three times with centrifugation at 500× g for 5 min at 4 °C.
  4. Add 1 mL of RPS to the cytoplasmic fraction and incubate at -20 °C for at least 1 h.
  5. Vortex the cytoplasmic fraction in RPS and centrifuge at 18,000× g for 15 min at 4 °C.
  6. Discard the supernatant and rinse the pellet with 70% (v/v) ice-cold ethanol.
  7. Add 1 mL of TRIzol to the semi-dry nuclear and cytoplasmic pellets followed by the addition of 10 μL of 0.5 M EDTA.
  8. Heat both fractions at 65 °C until the pellet dissolves with vortexing.
  9. Cool the samples to room temperature and add 200 μL of chloroform:isoamyl alcohol (1:24).
  10. Vortex the samples and centrifuge at 18,000× g for 10 min at room temperature.
  11. Transfer the aqueous supernatant into a clean microcentrifuge tube.
  12. Add an equal volume of isopropanol and incubate at -20 °C for at least 1 h.
  13. Vortex the samples and centrifuge at 18,000× g for 15 min at room temperature.
  14. Wash the pellet with 70% (v/v) ethanol and centrifuge at 18,000× g for 5 min.
  15. Air-dry the pellet for 5–10 min.
  16. Dissolve the air-dried RNA pellet in 30 μL of RNase-free water.
  17. Quantify the RNA concentration and purity with NanoDrop 2000c UV-Vis spectrophotometer.

cDNA synthesis

DNase I treatment for isolated RNA
1. Prepare the RNA in PCR tube for DNase I treatment as shown in Table 1.
  
  Table 1.Components used for DNase I treatment
  Components Stock concentration Final concentration Volume (μL)
  MuLV RT Buffer 10× 1× 1.4
  DNase I 1 U 0.5
  RNA 500–2,000 ng x
  H₂O Top up to 14
2. Incubate the tube in a thermal cycler at 37 °C for 30 min.

Reverse transcription (RT)

Prepare the RT reaction as shown in Table 2.

Table 2. Components used for reverse transcription

Components	Stock concentration	Final concentration	Volume (μL)
Components	Stock concentration	Final concentration	Random hexamer	Primer specific
MuLV RT Buffer	10×	1×	0.6	0.6
dNTP mix	10 mM	0.5 mM	1	1
Random hexamer	50 mM	2.5 mM	1	−
Reverse primers	10 μM	0.5 μM	−	0.5 for each primer
RNase inhibitor		2 U	0.5	0.5
Reverse transcriptase		10 U	0.5	0.5
H₂O			2.4	Top up to 6
Subtotal			6	6
DNAse I–treated RNA			14	14
Total			20	20

Perform the cDNA conversion on a thermal cycler with the setup of 42 °C for 60 min and follow by heat inactivation at 65 °C for 20 min.

Notes:
i. If more than four pairs of primer sets (targeting linear mRNAs and/or circRNAs) are needed for primer-specific cDNA conversion, prepare 5 μM primer mix and add 1 μL into the RT reaction.
ii. One may start with random hexamer for the cDNA conversion and switch to primer-specific cDNA conversion if the amplification of the targeted circRNA BSJs is weak, absent, or unspecific.
iii. Random hexamer-generated cDNA allows amplification of any circRNA target of interest at any one time, while primer-specific-generated cDNA only allows amplification of the planned circRNA targets and does not allow any amplification of any unplanned circRNA targets.
iv. Caution is needed when generating circRNA-specific cDNA, as the remaining unused reverse primer in the RT reaction could possibly interrupt or participate in qPCR process and lead to undesired amplification from linear mRNA.

Quantitative PCR (qPCR)
1. Prepare the PCR reaction as shown in Table 3.
  
  Table 3.Components used for SYBR Green RT-qPCR
  Components Stock concentration Final concentration Volume (μL)
  SYBR Fast qPCR Master Mix 2× 1× 5
  Forward primer 10 μM 0.1 μM 0.1
  Reverse primer 10 μM 0.1 μM 0.1
  6-fold diluted cDNA 3
  RNase-free water 1.8
  Total 10
2. Perform the qPCR on a real time thermal cycler with the cycling parameter of 95 °C for 3 min, followed by 40 cycles of 95 °C for 2 s and 60 °C for 20 s.
3. Calculate the relative expression of each gene based on the equation below:
  
  RNA ratio is the ratio of cytoplasmic RNA concentration to nuclear RNA concentration eluted in a similar volume of water. Gene expression for a cytoplasmic marker is calculated using formula (a), whereas gene expression of a nuclear marker is calculated using formula (b).

Data analysis

Representative data

Subcellular fractionation was performed using GM12878 cells, which is a lymphoblastoid cell line that contains Epstein-Barr Virus (EBV). The concentration and purity of each fraction were measured using NanoDrop 2000c (Table 4). The cytoplasmic RPL30 and nuclear MALAT1 transcripts were used as positive controls to indicate the purity of cytoplasmic and nuclear fractions, respectively. As shown in Figure 2, RPL30 was enriched in the cytoplasm, whereas MALAT1 was enriched in the nuclear fraction, indicating that subcellular fractionation was successfully performed. In this representative data, the linear EBV latent membrane protein 2 (LMP-2) transcripts were found in both cytoplasmic and nuclear fractions. The subcellular localization pattern of circLMP-2_e5 is similar to that of linear LMP-2A and LMP-2B.

Note that the protocol described here requires a real-time PCR machine to generate the data, which may not be available in all laboratories. In such a case, conventional agarose gel–based RT-PCR can be conducted to obtain a semi-quantitative result.

Table 4. Concentration and purity of cytoplasmic and nuclear fraction

	Concentration (ng/μL)	260/280	260/230
Cytoplasmic fraction 1	1,279	1.96	1.95
Cytoplasmic fraction 2	1,242	1.93	1.74
Nuclear fraction 1	566	1.76	0.71
Nuclear fraction 2	430.3	1.87	1.46

1 and 2 represent biological replicates.

Figure 2. Subcellular localization of circLMP-2_e5. RT-qPCR analysis showed LMP-2 and circLMP-2_e5 were localized in both nucleus and cytoplasm of GM12878 cells. Data was normalized to the RNA yield ratio and represents the mean ± SD of two independent experiments. This result was published in Figure 3C in Tan et al. (2021).

Validation of protocol

This protocol or parts of it has been used and validated in the following research article:

Tan et al. (2021). Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene. Scientific Reports (Figure 3, panel C).

Acknowledgments

The work was supported financially by the Ministry of Higher Education Malaysia via Fundamental Research Grant Scheme (FRGS/1/2017/SKK08/UM/02/11) and University of Malaya High Impact Research Grant (UM.C/625/1/HIR/MOE/CHAN/02/07).

The original research paper in which this protocol was described and validated is Tan et al. (2021).

Competing interests

The authors declare no competing interests.

References

Bustin S. A. and Nolan, T. (2004). Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15(3): 155–166.
Cai, Z., Fan, Y., Zhang, Z., Lu, C., Zhu, Z., Jiang, T., Shan, T. and Peng, Y. (2020). VirusCircBase: a database of virus circular RNAs. Briefings Bioinf. 22(2): 2182–2190.
Gagnon, K. T., Li, L., Janowski, B. A. and Corey, D. R. (2014). Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat. Protoc. 9(9): 2045–2060.
Jeck, W. R. and Sharpless, N. E. (2014). Detecting and characterizing circular RNAs. Nat. Biotechnol. 32(5): 453–461.
Szabo, L. and Salzman, J. (2016). Detecting circular RNAs: bioinformatic and experimental challenges. Nat. Rev. Genet. 17(11): 679–692.
Tan KE, Ng WL, Marinov GK, Yu KH, Tan LP, Liau ES, Goh SY, Yeo KS, Yip KY, Lo KW et al. (2021). Identification and characterization of a novel Epstein-Barr Virus-encoded circular RNA from LMP-2 Gene. Sci Rep. 2021 Jul 13;11(1):14392.
Zhang, Y., Yang, L. and Chen, L. L. (2016). Characterization of Circular RNAs. In: Feng, Y. and Zhang, L. (Eds.). Long Non-Coding RNAs (pp. 215–227). Methods in Molecular Biology. Humana Press, New York.