Materials and Reagents

1.5 ml microcentrifuge tubes (Quality Scientific Plastics, catalog number: 509-GRD-Q)
0.22 μm filter (Merk, Millex-GP PES, catalog number: SLGP033RB)
Nuclease-free pipette tips (Quality Scientific Plastics, catalog numbers: T104RLS-Q, T090RLS-Q and T112NXLRLS-Q)
Tris (AMRESCO, catalog number: 0497-5KG)
Sodium dodecyl sulfate (SDS) (AMRESCO, catalog number: 0227-1KG)
EDTA, disodium salt, dihydrate (Na₂EDTA·2H₂O) (AMRESCO, catalog number: 0105-1KG)
Proteinase K (AMRESCO, catalog number: 0706-100MG)
6x DNA Loading Dye (Thermo Fisher Scientific, catalog number: R0611)
Agarose (BIOWEST, Regular Agarose G-10, catalog number: 111860)
Nuclease-free water (AMRESCO, catalog number: E476-1L)
Double distilled water (ddH₂O)
DNase I, RNase-free, supplied with 10x Reaction buffer with MgCl₂ (Thermo Fisher Scientific, catalog number: EN0521)
TRIzol (Life Technologies, catalog number: 15596018)
1-Bromo-3-chloropropane (Sigma-Aldrich, catalog number: B9673)
Isopropanol (AMRESCO, catalog number: 0918-1L)
Ethanol (Sigma-Aldrich, catalog number: 459836-2L)
HCl (Beijing Chemical Works, Analytic Reagent grade)
NaOH (sodium hydroxide pellets) (Shanghai Sangon Biotech, catalog number: A100173)
GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific, catalog number: SM0311)
HEPES free acid (AMRESCO, catalog number: 0511-1KG)
Mannitol (AMRESCO, catalog number: 0122-500G)
Sucrose (AMRESCO, catalog number: 0335-500G)
MitoPrep buffer (see Recipes)
10% (w/v) SDS (see Recipes)
1 M Tris-HCl (pH 7.4) (see Recipes)
0.5 M EDTA (pH 8.0) (see Recipes)
Proteinase K (1 mg/ml) (see Recipes)
Lysis buffer (see Recipes)
1x DNase buffer (see Recipes)
75% ethanol (v/v) (see Recipes)

Equipment

Pipettes (RAININ, Pipet-Lite XLS)
Two heat blocks (Hangzhou Allsheng Instruments, Product Name: dry bath incubator, catalog number: MK200-2)
NanoDrop instrument (Thermo Fisher Scientific, NanoDrop 2000c Spectrophotometer)
Power supply (Tanon, catalog number: EPS 300)
Gel imaging system (Tanon, catalog number: 1600)
Centrifuges (Thermo Fisher Scientific, models: Sorvall Legend Micro 21 and Micro 21R)
pH meter (Sartorius, catalog number: PB-10)
-80 °C freezer (Thermo Fisher Scientific)
Magnetic stir bars and magnetic stirrer
Fume hood
Laminar flow cabinet
Incubator
4 °C refrigerator
-20 °C freezer

Procedure

The flow sheet of this protocol is shown in Figure 1.

Figure 1. The flow sheet of steps in this protocol

Sample pretreatment
Note: To examine sub-mitochondrially localized RNA, different treatments such as nuclease treatment or mitoplasting are needed. We focus here on inactivation of mitochondrial RNases. Other methods such as proximity labeling are also needed to separate mitochondrially localized RNA from RNA contamination from other cellular compartments.
1. Isolate mitochondria from tissue culture cells as described in Huang and Wang (2019) and measure mitochondrial protein concentration with a NanoDrop instrument after diluting 1 μl of mitochondrial sample with 19 μl of 0.6% SDS. Transfer 200 μg of purified mitochondria (in 20 μl of MitoPrep buffer) to a microcentrifuge tube and keep the tube on ice.
  Note: About 9 x 10⁶ HEK293 cells could be harvested from a 10-cm cell culture dish of 90% cell density, for a yield of around 250 μg mitochondria.
2. Mix mitochondria with 100 μl of fresh lysis buffer and heat the sample at 70 °C in a heat block for 5 min. Take out the sample, and let it cool down to room temperature (RT).
3. Add 1 μl of 1 mg/ml proteinase K to the lysate and incubate it at 37 °C in a heat block for 5 min.
RNA extraction
Note: Perform this part in a fume hood.
1. Add 400 μl of TRIzol and 200 μl of 1-Bromo-3-chloropropane to the pretreated lysate, shake the tube hard for 1 min, and then spin at 14,800 x g for 5 min at RT.
2. Transfer the colorless fraction in the upper layer to a new microcentrifuge tube, mix with an equal volume of isopropanol and spin at 21,000 x g for 10 min at 4 °C.
3. Discard the supernatant and save the RNA pellet. Add 600 μl of ice-cold 75% ethanol to the tube, gently turn it upside down for a couple of times, and spin at 21, 000 x g for 5 min at 4 °C.
  Note: If a small amount of mitochondria (200 μg or less) was used, the RNA pellet might be very small and hardly visible.
4. Discard the supernatant and make the remaining ethanol as little as possible. Be careful to avoid losing the pellet.
5. Dry the RNA pellet in the laminar flow cabinet.
RNA treatment
Note: If mitochondrial DNA would not interfere with downstream assays, DNase I treatment is not required and RNA pellets can be directly dissolved in nuclease-free water.
1. Dissolve the RNA pellet in 40 μl of 1x DNase buffer with 0.5 μl of DNase I, incubate it at 37 °C for 10 min, vortex the tube, and then spin down the sample briefly. Incubate the sample again at 37 °C for 10 min.
2. Add 0.4 μl of 0.5 M EDTA to the RNA sample and heat the sample at 70 °C in a heat block for 10 min to inactivate the DNase I.

Data analysis

Representative data (Figure 2)

Figure 2. Agarose gel image of the isolated mitochondrial RNA, which was treated with or without DNase I. Three hundred nanograms of the isolated RNA were treated with (+DNase I) or without (-DNase I) DNase I. After DNase I inactivation, RNA samples were separated on a 1.5% agarose gel stained with ethidium bromide.

Data processing
The gel image was captured with Tanon 1600 Gel Image System (Tanon), and cropped using Photoshop.

Data analysis
No statistical analysis was performed.

Recipes

MitoPrep buffer
0.225 M mannitol
0.075 M sucrose
20 mM HEPES (pH 7.4)
Dissolve 2.05 g of mannitol (Mw: 182.17), 1.28 g of sucrose (Mw: 342.3), and 0.2383 g of HEPES (Mw: 238.3) in ~45 ml ddH₂O, adjust the pH to 7.4 with 1 M KOH, and add ddH₂O to the final volume of 50 ml
Filter through a 0.22 μm filter, and store at 4 °C
10% (w/v) SDS
Dissolve 10 g of SDS in 100 ml of ddH₂O and stir until SDS is completely dissolved
Store at RT. Dilute 10% SDS to 0.6% SDS with ddH₂O
1 M Tris-HCl (pH 7.4)
Dissolve 12.114 g of Tris (Mw: 121.14) in ~80 ml of ddH₂O, stir until completely dissolved
Adjust the pH to 7.4 with HCl, and make up to 100 ml with ddH₂O
Filter through a 0.22 μm filter and store at RT
0.5 M EDTA (pH 8.0)
Add 9.306 g of EDTA (Mw: 372.24) to ~40 ml of ddH₂O, stir and slowly adjust the pH to 8.0 with NaOH, and make up to 50 ml with ddH₂O
Filter through a 0.22 μm filter and store at 4 °C
Note: EDTA will not fully dissolve until the pH is close to 8.0.
Proteinase K (1 mg/ml)
Dissolve 1 mg of proteinase K in 1 ml of nuclease-free water, aliquot and store at -20 °C
Lysis buffer
The lysis buffer consists of 1% SDS, 10 mM EDTA and 10 mM Tris-HCl (pH 7.4)
For a 10 ml solution:
Mix 8.7 ml of nuclease-free water, 1 ml of 10% SDS, 0.2 ml of 0.5 M EDTA (pH 8.0), and 0.1 ml of 1 M Tris-HCl (pH 7.4) and make up to 10 ml with nuclease-free water
1x DNase buffer
Dilute 10x DNase buffer (with MgCl₂) to 1x with nuclease-free water
75% ethanol (v/v)
For a 100 ml solution, add 25 ml of nuclease-free water to 75 ml of ethanol
Mix thoroughly and store at 4 °C

Acknowledgments

This work was supported by Grant 2017YFA0504600 from the Priority Research Program of the Ministry of Science and Technology of China, by Grants 31371439 and 91649103 from the National Natural Science Foundation of the People’s Republic of China, and by funds from the Ministry of Education of the People’s Republic of China 1000 Talents Youth Program. This protocol was modified from Huang et al. (2018) and Liu et al. (2017).

Competing interests

The authors declare no conflicts of interest with the contents of this article.

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