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Protocol for Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR

Experiment Summary

Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.

Materials and Reagents

  1. GM12878 cell line
  2. Dulbecco's phosphate-buffered saline (PBS)
  3. Trypsin-EDTA solution (0.5%)
  4. Tissue culture media
  5. Fetal bovine serum (FBS)
  6. Penicillin-Streptomycin
  7. Trypan blue
  8. Tris-Base
  9. Potassium chloride (KCl)
  10. Magnesium chloride (MgCl2)
  11. DL-Dithiothreitol (DTT)
  12. NP-40
  13. Ribonucleoside vanadyl complexes
  14. Sodium acetate
  15. Molecular grade ethanol
  16. TRIzol
  17. Chloroform:isoamyl alcohol (1:24)
  18. Molecular grade isopropanol
  19. RNase-free water
  20. 10× M-MuLV RT buffer
  21. DNase I
  22. M-MuLV reverse transcriptase
  23. RNase inhibitor
  24. dNTP mix
  25. Random hexamer
  26. Primers
  27. SYBR Fast qPCR Master Mix

Equipment

  1. Refrigerated tabletop centrifuge (15 or 50 mL conical tube adaptors)
  2. Refrigerated benchtop centrifuge (1.5 mL tube rotor)
  3. Light microscope
  4. Vortexer
  5. Micropipettors
  6. 2000c UV-Vis Spectrophotometer
  7. PCR Thermocycler
  8. Real-Time PCR System

Procedure

A. Subcellular fractionation

1. Cell harvesting from culture plates

a) Grow the cells to a confluency of ~80%. Rinse the cells with 1× PBS.

b) For adherent cells, detach the cells with Trypsin-EDTA. For suspension cells, proceed to step A1d.

c) Add an equal volume of complete media to stop the trypsin proteolysis.

d) Collect the cell suspension in a conical tube and centrifuge at 200× g for 5 min.

e) Remove the supernatant and resuspend the cell pellet with 1× PBS.

f) Perform cell counting and aliquot 1 × 106 cells into a 1.5 mL microcentrifuge tube.

g) Centrifuge the tube at 200× g for 5 min and remove the supernatant.

h) Keep the cell pellet on ice prior to the next step.

2. RNA isolation from cytoplasmic and nuclear fractions of cells

a) Resuspend the cell pellet gently with 100 μL of ice-cold hypotonic buffer and incubate on ice for 5 min.

b) 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.

c) 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.

d) Add 1 mL of RPS to the cytoplasmic fraction and incubate at -20 °C for at least 1 h.

e) Vortex the cytoplasmic fraction in RPS and centrifuge at 18,000× g for 15 min at 4 °C.

f) Discard the supernatant and rinse the pellet with 70% (v/v) ice-cold ethanol.

g) 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.

h) Heat both fractions at 65 °C until the pellet dissolves with vortexing.

i) Cool the samples to room temperature and add 200 μL of chloroform:isoamyl alcohol (1:24).

j) Vortex the samples and centrifuge at 18,000× g for 10 min at room temperature.

k) Transfer the aqueous supernatant into a clean microcentrifuge tube.

l) Add an equal volume of isopropanol and incubate at -20 °C for at least 1 h.

m) Vortex the samples and centrifuge at 18,000× g for 15 min at room temperature.

n) Wash the pellet with 70% (v/v) ethanol and centrifuge at 18,000× g for 5 min.

o) Air-dry the pellet for 5-10 min.

p) Dissolve the air-dried RNA pellet in 30 μL of RNase-free water.

q) Quantify the RNA concentration and purity with NanoDrop 2000c UV-Vis spectrophotometer.

B. cDNA synthesis

1. DNase I treatment for isolated RNA

a) Prepare the RNA in PCR tube for DNase I treatment as shown in Table 1.

Table 1. Components used for DNase I treatment

ComponentsStock concentrationFinal concentrationVolume (μL)
MuLV RT Buffer10×1×1.4
DNase I1 U0.5
RNA500-2,000 ngx
H2OTop up to 14

b) Incubate the tube in a thermal cycler at 37 °C for 30 min.

2. Reverse transcription (RT)

a) Prepare the RT reaction as shown in Table 2.

Table 2. Components used for reverse transcription

ComponentsStock concentrationFinal concentrationVolume (μL)
Random hexamerPrimer specific
MuLV RT Buffer10×1×0.60.6
dNTP mix10 mM0.5 mM11
Random hexamer50 mM2.5 mM1−
Reverse primers10 μM0.5 μM−0.5 for each primer
RNase inhibitor2 U0.50.5
Reverse transcriptase10 U0.50.5
H2O2.4Top up to 6
Subtotal66
DNAse I-treated RNA1414
Total2020

b) 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.

C. Quantitative PCR (qPCR)

1. Prepare the PCR reaction as shown in Table 3.

Table 3. Components used for SYBR Green RT-qPCR

ComponentsStock concentrationFinal concentrationVolume (μL)
SYBR Fast qPCR Master Mix2×1×5
Forward primer10 μM0.1 μM0.1
Reverse primer10 μM0.1 μM0.1
6-fold diluted cDNA3
RNase-free water1.8
Total10

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:

Protocol for Detection of Cytoplasmic and Nuclear Circular RNA via RT-qPCR

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).

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* For research use only. Not intended for any clinical use.
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