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Isolation of highly enriched mitochondrial DNA from mouse tissues

The protocol has been mainly optimized for soft tissues, such as liver and brain (as in [11]). However, it also works for heart, although more extensive yet gentle homogenization is required. The mtDNA yield from heart is also much lower than from the bigger tissues and nDNA contamination levels are often higher

REAGENT SETUP

CRITICAL: Reagent storage is at room temperature unless mentioned otherwise.

STOCK SOLUTIONS

• Acetic acid, 2 M Combine 11.48 ml glacial acetic acid with 88.52 ml distilled H2O to obtain 100 ml 2 M acetic acid solution.

• DNase I, 5 mg/ml in 5 mM CaCl2 Dissolve DNase I according to the weight into suitable volume of 5 mM CaCl2 (diluted from 1 M CaCl2 stock solution, e.g. 7.5 µl 1 M CaCl2 in 1.5 ml distilled H2O) to obtain 5 mg/ml DNase I solution. Dispense into aliquots and store at -20 °C.

CRITICAL: DNase I activity might decrease if the storage period exceeds several months.

• dNTPs, 1.25 mM each Combine 250 µl of each dNTP (100 mM stocks) and add 19 ml distilled H2O to obtain a solution containing 1.25 mM each dNTP. Dispense into aliquots and store at -20 °C.

EDTA, 0.5 M, pH 8.0 Dissolve 18.1 g EDTA into 80 ml distilled H2O. Adjust the pH to 8.0 with NaOH and bring the volume to 100 ml with distilled H2O.

• EtOH, 70-% Measure 35 ml of absolute EtOH and 15 ml distilled H2O separately and combine to obtain ca. 48 ml of 70-% EtOH.

CRITICAL: 70-% EtOH is hygroscopic i.e. it will evaporate and absorb water over time, which will lower the concentration. Thus, always use rather freshly prepared 70-% EtOH solution.

• NaOH, 15 M Dissolve 600 g NaOH into 600 ml distilled H2O. Bring the volume to 1 liter with distilled H2O.

CAUTION! The reaction is exothermic. The solution can be kept on ice during preparation.

• Potassium acetate, 6 M Dissolve 58.92 g potassium acetate into 40 ml distilled H2O. Adjust the pH to 7.5 with 2 M acetic acid. Bring the volume to 100 ml with H2O.

CRITICAL: Long-term storage is recommended at -20 °C.

• Proteinase K, 10 mg/ml Dissolve 100 mg Proteinase K into 10 ml of distilled H2O. Dispense into aliquots and store at -20 °C.

RNase A (free of DNase), 9.09 mg/ml Dissolve RNase A at a concentration of 10 mg/ml into 10 mM sodium acetate pH 5.2 (e.g. dissolve 100 mg of RNase A into 1 ml of distilled H2O, and dilute 100 µl of that RNase A solution with 890 µl of distilled H2O and 10 µl of 1 M sodium acetate). Incubate the aliquot tubes in a heat block at 100 °C for 15 min. Allow the tubes to slowly cool down to room temperature. Adjust the pH by adding 100 µl (i.e. 0.1 volumes) of 1 M Tris (pH 7.4) to obtain 9.09 mg/ml RNase A solution. Dispense into aliquots and store at -20 °C.

CRITICAL: RNase A precipitates if the concentrated solution is boiled at neutral pH.

• SDS, 10-% Dissolve 10 g SDS into 80 ml distilled H2O. Bring the volume to 100 ml with distilled H2O.

• Sodium acetate, 1 M, pH 5.2 Dissolve 24.61 g sodium acetate into 50 ml distilled H2O. Adjust the pH to 5.2 with glacial acetic acid. Allow the solution to cool down to room temperature and adjust the pH again to 5.2 with glacial acetic acid. Bring the volume to 100 ml with distilled H2O.

• Tris, 1 M, pH 7.4 Dissolve 12.11 g Tris into 80 ml distilled H2O. Adjust the pH to 7.4 with HCl. Allow the solution to cool down to room temperature and adjust the pH again with HCl. Bring the volume to 100 ml with distilled H2O.

• Tris, 1 M, pH 8.0 Prepare as Tris, 1 M, pH 7.4, but adjust the pH to 8.0.

BUFFERS

• Lysis buffer: 20 mM Tris, 150 mM NaCl, 20 mM EDTA, 1 % SDS, pH 8.75 Dissolve 0.877 g NaCl into 60 ml distilled H2O. Add 2 ml Tris (1 M, pH 8.0), 4 ml EDTA (0.5 M, pH 8.0) and 10 ml SDS (10-%). Adjust the pH to 8.75 with NaOH at room temperature. Bring the volume to 100 ml with distilled H2O.

CRITICAL: High concentrations of NaCl and SDS precipitate.

CRITICAL: Tris pH is temperature dependent: pH 8.75 at room temperature corresponds to pH 7.8 at the usage temperature of 56 °C.

• Mitochondria isolation buffer (MIB), 2x concentrate: 640 mM sucrose, 40 mM Tris, 2 mM EGTA, pH 7.2 Dissolve 219 g sucrose, 4.58 g Tris, 0.76 g EGTA into 950 ml distilled H2O. Adjust the pH to 7.2 with HCl and bring the solution to 1 liter with distilled H2O. Aliquot and store the 2x concentrate MIB at -20 ℃ until use.

CRITICAL: Tris pH is temperature dependent: pH 7.2 at room temperature corresponds to pH ca. 7.8 at the usage temperature of 0-4 ℃.

CRITICAL: MIB 2x concentrate solution can be stored frozen for very long periods of time (>12 months).

• Mitochondria isolation buffer (MIB02): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 0.2 % BSA, pH 7.2 Thaw 2x concentrate MIB and dilute it with equal volume of distilled H2O to obtain 1x MIB solution. Add 0.2 % (w/v) BSA to obtain MIB02 solution.

CRITICAL: Store thawed MIB at 4 ℃ only short periods of time (1-2 days).

CRITICAL: BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.

Mitochondria isolation buffer (MIB1): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 1 % BSA, pH 7.2 Thaw 2x concentrate MIB and dilute it with equal volume of distilled H2O to obtain 1x MIB solution. Add 1 % (w/v) BSA to obtain MIB1 solution.

CRITICAL: Store thawed MIB at 4 ℃ only short periods of time (1-2 days).

CRITICAL: BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.

• Mito-DNase buffer base: 300 mM sucrose, 10 mM MgCl~2~, 20 mM Tris, pH 7.5 Dissolve 10.27 g sucrose, 0.203 g MgCl2 and 0.243 g Tris into 90 ml distilled H2O. Adjust the pH to 7.5 with HCl and bring the solution to 100 ml with distilled H2O. Aliquot and store the Mito-DNase buffer base at -20 ℃ until use.

CRITICAL: Tris pH is temperature dependent: pH 7.5 at room temperature corresponds to pH ca. 7.2 and 8.1 at the usage temperatures of 37 and 0-4 ℃, respectively.

• Mito-DNase buffer: Mito-DNase buffer base, 0.15 % (w/v) BSA, 0.03 mg/ml DNase I, 0.02 mg/ml RNase A Thaw Mito-DNase buffer base and add 0.15 % (w/v) BSA. Just before use, add 0.03 and 0.02 mg/ml DNase I and RNase A, respectively.

CRITICAL: BSA-containing Mito-DNase solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.

EQUIPMENT

• 1.5-ml microcentrifuge tubes

• 250-µl low-bind, wide-orifice pipette tips (VWR, cat. no. 613-0370)

• 50-ml polypropylene Falcon tubes

• Gel electrophoresis system (e.g. Bio-Rad Sub-Cell(TM) GT Cell)

• Gel imaging system (e.g. Syngene U:Genius)

• Heat block for 1.5 ml microcentrifuge tubes (e.g. Grant QB-H2)

• Motor-driven glass/Teflon Potter Elvehjem homogenizer (e.g. Sartorius Potter S)

• PCR tubes (e.g. VWR® 8-well tube strips with bubble cap)

• PCR thermocycler (e.g. The Applied Biosystems® Veriti® 96-Well Thermal Cycler)

• Qubit® Assay tubes (Thermo Fisher Scientific, cat.no. Q32856)

• Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, cat.no. Q33216)

• Refrigerated centrifuge for 1.5 ml microcentrifuge tubes (e.g. Eppendorf 5417R)

• Refrigerated centrifuge for 50-ml Falcon tubes, swing- and fixed rotors (e.g. Eppendorf 5804R)

Tissue collection and homogenization (1-1.5 hour)

1-Sacrifice a mouse and collect 500-800 mg of liver tissue or a full brain (ca. 430 mg). Rinse the tissue in ice cold PBS. Store the tissue in a 50-ml Falcon tube in 5-10 ml ice cold PBS until homogenization.

NOTEUse only freshly collected tissue for mitochondria isolation.

2- Homogenize the tissue with a teflon pestle by 5 strokes with 200 rpm. (Experimental: Homogenization of heart tissue requires harsher conditions.)

NoteAvoid breakage of the nucleus or mitochondria by too rigorous homogenization.

3- Remove PBS and transfer the tissue into glass homogenizer tube with 15 ml ice cold MIB1.

4-Transfer the homogenate back to the 50-ml Falcon tube and add 15 ml ice cold MIB1

Mitochondria isolation (~1 hour)

5-Remove cell debris by centrifugation at 800 g 4 ℃ for 10 min.

4 °C 800g

00:10:00

6-Pour the supernatant into a clean 50-ml Falcon tube and repeat the centrifugation.

NOTEIn case a fat layer is formed on top of the liquid, dip a pipet tip gently into the solution and remove as much fat as possible.

00:10:00

4 °C 800g

7-Pour the supernatant carefully into a clean 50-ml Falcon tube.

NOTEDo not disturb the pellet.

8-Collect mitochondria by centrifugation at 8,500 g 4 ℃ for 10 min.

00:10:00

4 °C 8500g

9- discard supernatant by pouring and carefully pipet out all leftover liquid. Continue to the next step immediately.

Nuclear DNA removal (~3 hours)

10- Add 0.03 mg/ml DNase I and 0.02 mg/ml RNase A into ice cold Mito-DNase buffer.

11- Dissolve mitochondria pellet carefully into 600 µl Mito-DNase buffer by pipetting up and down. Keep the tubes on ice.

NOTEUse wide-orifice pipet tips when pipetting mitochondria-containing solutions.

NOTEHarsh pipetting will break mitochondria and decrease the overall yield, but thorough dissolving is required for DNase I to access and digest any nDNA leftovers increasing the purity of the final mtDNA sample.

12- Divide dissolved mitochondria solution into subfractions: the number of fractions depend on the input tissue amount such that each fraction represents ca. 100-150 mg tissue in 600 µl Mito-DNase buffer. For example, for a full brain tissue (ca. 430 mg) add 2x 600 µl Mito-DNase buffer (final volume 1.8 ml), mix well by pipetting and divide the solution into three 1.5-ml microcentrifuge tubes.

NOTEUse wide-orifice pipet tips when pipetting mitochondria-containing solutions.

13- Incubate the tubes at 37 ℃ for 1-1.5 hours.

01:00:00

37 °C

14- Collect mitochondria by centrifugation at 13,000 g 4 ℃ for 30 min.

00:30:00

13000g

15- Remove supernatant thoroughly.

16- Wash out the leftovers of Mito-DNase buffer by carefully re-suspending the mitochondria pellet into 500 µl MIB02, and re-pelleting by centrifugation at 13,000 g 4 ℃ for 15 min.

00:15:00 4 °C

13000g

17- Repeat the previous wash step.

00:15:00 4 °C

13000g

18- Remove the supernatant, spin down the leftover liquid and pipet out any traces of liquid.

19-Freeze the pellet in liquid nitrogen and store at -80 ℃ until DNA extraction.

Note

Mitochondria pellets can be stored at -80 ℃ at least a few hours. Longer storage times (weeks/months) is not expected to affect the mtDNA extraction, but has not been tested during this protocol optimization.

DNA extraction (~3-20 hours)

20- Add 0.02 mg/ml Proteinase K and 0.02 mg/ml RNase A into lysis buffer preheated to 56 ℃.

56 °C

21-Dissolve frozen mitochondria pellets into 400 µl lysis buffer by pipetting up and down vigorously.

22-Incubate the tubes at 56 ℃ overnight (ca. 14-16 hours).

16:00:00

56 °C

NOTEShorter lysis time can be used, even 1-hour lysis should be sufficient, but has not been tested during this protocol optimization.

23-Let the solution cool down to room temperature

24- Add 100 µl potassium acetate.NoteWhite precipitate forms when potassium reacts with SDS.

25-Add 500 µl of chloroform:isoamylalcohol (24:1) and shake the tubes for 20 s. 00:00:20

26-Separate the phases by centrifugation at 16,000 g at room temperature for 10 min.00:10:00 22 °C 16000g

27-Transfer the upper aqueous phase carefully to a clean 1.5-ml microcentrifuge tube.

28-Add 100 or 200 µg of RNase A (i.e. 11 or 22 µl for brain or liver sample, respectively).

Note Complete removal of RNase A might require additional chloroform : isoamylalchol purification step instead of EtOH precipitation alone. However, additional purification step may decrease the final yield and is not necessarily required for a successful Illumina sequencing.

29- Incubate the tubes at 37 ℃ for 45-60 min.

00:45:00 37 °C

30-Add 1 ml (2 volumes) ice cold absolute EtOH and invert the tubes gently for 5 times.NoteIf, for example, smaller amount of tissue than recommended in this protocol is used and low concentration of DNA expected, glycogen can be used as a carrier to increase the DNA yield in EtOH precipitation. Even glycogen with a dye (e.g. Glycoblue) is compatible with Illumina sequencing.

31-Incubate the tubes at -80 ℃ at least for 30 min.03:00:00 -80 °C NoteIncubation can be even 2-3 hours. Alternatively, prolonged (e.g. overnight) incubation at -20 ℃ is possible.

32-Pellet DNA at 16,000 g at room temperature for 15 min.

00:15:00 22 °C 16000g

33-Remove supernatant by pipetting and wash DNA with 500 µl 70 % EtOH. Pellet DNA by centrifugation at 16,000 g at room temperature for 5 min

.00:05:00 22 °C 16000g

34-Repeat the wash step in order to increase the purity of the DNA.00:05:00 22 °C 16000g NoteDo not disturb the pellet.

35-After the last wash, spin down the leftover EtOH and remove all traces by pipetting.

36- Air-dry the pellet briefly (1-2 min) until all trace ethanol has evaporated.

Note Over-drying the DNA might lead to a non-dissolving pellet or damage the DNA.Drying the pellet with heat is not recommended as higher temperatures may also damage the DNA.

37-Dissolve the DNA pellet by adding 18-30 µl elution buffer AE (5 mM Tris/HCl, pH 8.5) depending on the DNA pellet size and required final concentration. Incubate the tubes at room temperature overnight.16:00:00

Note Dissolving the pellet with heat is not recommended as higher temperatures may damage the DNA. Note DNA is less stable in H~2~O and pH of H~2~O might not be optimal for DNA storage, whereas EDTA of TE buffer might not be suitable for downstream applications such as Illumina sequencing. Note High yield of mtDNA might appear as a brown pellet and brown color might be visible also in the dissolved DNA solution. This affects spectrophotometric DNA concentration measurement, thus, fluorometric method should be used in order to have more reliable concentration measurement.

38- Short-term storage is recommended at 4 ℃, long-term storage at -20 ℃.

Note Multiple freeze-thaw cycles should be avoided. Note Continue to the next step earliest on the following day of dissolving the DNA, or stop the protocol here and store the DNA at 4 or -20 ℃ according to the length of the pause. However, it is recommended to continue with the quality control measurements and combination of the final samples before long-term storage of the DNA sample at -20 ℃ in order to avoid unnecessary freeze-thaw cycles.

39-DNA quality control (~3-4 hours)39Measure the concentration of 1 µl sample with Qubit HS kit twice and take the average as the sample concentration. If the sample is too concentrated, remeasure with suitable dilution.

40-Test for nDNA contamination by performing 20-µl PCR reaction with primers designed for an nDNA-encoded target (here, we use primers specific for PolgA (expected product size 520 bp).

Master mix for a single 20-µl PCR reaction:

µl Reagent Final concentration

28.9 H2O adjust according to the sample volume

4.0 5x Green GoTaq reaction buffer 1x

3.2 dNTPs (1.25 mM each) 200 µM

1.2 MgCl2 (25 mM) 1.5 mM

0.8 PolgA_F (10 µM) 0.4 µM

0.8 PolgA_R (10 µM) 0.4 µM

0.1 GoTaq DNA polymerase (5u/µl) 0.5 units

1.0 mtDNA sample see note

PCR programme:

94 C - 60 s

---------------

30 cycles of:

94 C - 30 s

58 C - 30 s

72 C - 45 s

---------------

72 C - 3 min

8 C - Inf

Note Generally, 1 µl of the sample is suitable for the PCR, however, if sample concentrations are highly variable between samples, it is advisable to normalize the input DNA amounts.

Note Use genomic DNA, such as a quick tail extract, as a positive control.

Note For more accurate quality control, quantitative PCR is recommended as described by Kennedy et al. (2013) [11]

Note Heavy RNA contamination inhibits the PCR reaction and might give false-negative result.

41- Load the 20-µl PCR reaction fully on 1-% agarose gel in 0.5x TBE buffer containing 0.5 µg/ml EtBr. Only load 5 µl or less of the positive control sample

.Note No or only very slightly visible DNA band should be observed on the gel (see Figure 1A). nDNA contaminated fractions should not be used for further analysis. The isolation protocol should be optimized, often starting from the homogenization step, in order to reduce the nDNA contamination. Note Mitochondrial DNA enrichment can be verified by PCR reaction with primers specific for a mtDNA-encoded gene.

42-Combine fractions from the same tissue which show no or extremely minor nDNA contamination and re-measure the final sample concentration with Qubit HS. Dispense the sample into aliquots and store at -20 ℃.NoteIf higher (maximum double) concentration is needed for the downstream application, concentrate the sample by vacuum at 45 ℃.

43-Test the quality of the mtDNA sample by running 100 ng (according to the Qubit HS concentration measurement) on 1-% agarose gel.

Note Circular mtDNA shows multiple bands on the gel according to different forms: supercoiled plasmid, relaxed plasmid and/or linear DNA. There should not be strong smears.

NoteEspecially in a short gel run (ca. 30 min), RNA contamination can be seen as two distinct low-molecular weight bands, or more significant RNA contamination might also appear as a huge blob

Good Luck

Hello,

What volume of buffer AE did you elute in when using the QIAGEN Blood and Tissue kit? I use the kit to extract DNA from mouse ear punches and elute in 50ul AE, this usually gives a DNA concentration of 10-20ng/ul when measured on the nanodrop. If you tried the 200ul recommended in the QIAGEN protocol then it might be worth using a smaller elution volume.

What does your genotyping assay involve? If it is PCR-based then you will not need much input DNA at all, so you might not need very high DNA yield anyway.

Stephen

Hi Ying Wang - As for genotyping, what is your target and how large a piece do you need? We routinely genotype mtDNA relative heretoplasmy by pyrosequencing of specific heteroplasmic sites in mice on a very crude heat+base/neutralize protocol. As long as you PCR, it works very well (and is really cheap). You can message me here or find my e-mail at with more more details on what you are after, then I can post the protocol.

---

Some added info on the protocol Ahmed Housanie Elsappah posted...

That protocol was written up by my former PhD student Marita Isokallio;

https://www.protocols.io/view/isolation-of-high-quality-highly-enriched-mitochon-mycc7sw

Pg 76 of her thesis shows 4 tissues where we tried the protocol (https://kups.ub.uni-koeln.de/7941/1/Thesis_Isokallio_A5_twosided.pdf) It was optimized on soft tissues (liver, brain), and we have not tried it on tails. The "harder" the tissue (ie heart), the less well it worked. It was also quite bad in the spleen. This is probably not great for small tail pieces.

Thanks for all your answers! Much appreciated!

In addition to all the above suggestions, I should recommend trying plasmid miniprep kit for the same. I have tried it (considering mitogenome is nothing but slightly larger (than usual) plasmids) for enriching mtDNA in the DNA extracts and verified the results using qPCR, which suggested enrichment of mtDNA. I also found couple of papers based on similar principle of alkaline lysis (although they also involve use of paramagnetic beads as well).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353588/

https://link.springer.com/protocol/10.1007/978-1-4939-2257-4_9

I would consider a simpler approach. After Proteinase K treatment (just to be sure), addition of alkaline lysis buffer, followed by neutralization as per kit/plasmid miniprep protocol and silica column purification shall yield pure total DNA enriched with mtDNA.