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Denver Genetic Laboratories
 

Blue native electrophoresis with in-gel mitochondrial enzyme analysis

Clinical Test Information - UCD Biochemical Genetics Laboratory


 

CPT Code:

82664 x 1, 82657 x 4 (four assays are performed).

Availability:

Monday - Friday, 0800 - 1700.

Turnaround Time:

Fibroblasts: 2 3 months (depending on growth rate of cultured cells)
Muscle or Liver: One month (or six weeks if combined with mitochondrial respiratory chain enzyme assay).

Specimen Requirements:

  • Liver tissue: Minimum 50 mg required.
  • Muscle tissue: Please see the detailed instructions below. Minimum 60 mg required, 100 mg recommended. 
  • Skin fibroblasts: Two T-25 flasks of confluent fibroblasts, with plug-seal caps, completely filled with media. Fibroblasts must be Mycoplasma-free.

LIVER

Liver samples can be obtained as a wedge biopsy or as a needle biopsy. It is important to ensure that sufficient liver tissue is obtained. If using a needle biopsy, obtain two (2) needle biopsies 14 Gauche Bard monopty instrument 2 cm in length. Postmortem samples must be obtained within 6 hours of death, and preferably within 2 hours of death, as interpretation will become more difficult with time.

Place the liver biopsy in either Parafilm or aluminum foil, and then place in a 2 ml cryotube, then close the screw cap tightly.

Freeze immediately in liquid nitrogen or on dry ice, and maintain in frozen condition. If possible, weigh the liver biopsy before placing it into the cryotube. If the specimen was not weighed before it was frozen, DO NOT attempt to weigh the frozen sample as the samples degrade very easily. Instead, ship the sample to the RC Core Lab without a weight. Lab personnel will weigh the sample.

Keep sample in -70° C freezer. Samples will not be stable at -20° C. Samples that have thawed even for a short period are not acceptable. Weighing the sample will cause thawing. DO NOT ATTEMPT TO WEIGH A FROZEN SAMPLE.

MUSCLE

Muscle biopsies must be obtained without the use of electrocautery and must be completely processed within 5 to 10 minutes of being excised. Muscle biopsies should be preferably about 120 mg in size. They must be frozen immediately in liquid nitrogen or on dry ice. They cannot be placed in any preservative including OCT. They must be stored in a microvial at -70° C until shipment to the laboratory on dry ice.

All muscle tissue specimens should be frozen immediately in liquid nitrogen, or on dry ice, and stored at -70° C. Specimens should be shipped frozen on dry ice.

Please also enclose a completed requisition form for mitochondrial respiratory chain enzyme assays (please make sure that you enclose the appropriate requisition form, and that both pages of the requisition form are completed). Do not forget to fill out recent use of selected antibiotics on the requisition form as these may interfere with test interpretation.

FIBROBLASTS

Send flasks in a Styrofoam box at room temperature. Please include passage number (if known).

Please also enclose a completed requisition form for mitochondrial respiratory chain enzyme assays (please make sure that you enclose the appropriate requisition form, and that both pages of the requisition form are completed).

Primary contact for mitochondrial enzyme assay:

Marisa Friederich, PhD
Phone: 303-724-3841
Email: marisa.friederich@ucdenver.edu

Interpretive Reference Ranges:

Blue native gel results are qualitatively interpreted in references to a control specimen run at the same time. Interpretation will focus on intensity of the band and on the presence of abnormal bands suggesting incomplete assembly of the complex.

Use:

Diagnosis of mitochondrial respiratory chain diseases.

Methodology:

Isolation of mitochondrial membranes, gel electrophoresis, and in-gel specific enzyme activity staining.​