| Mice fed basal diet supplemented with 0 or 0.2 µg Se/g diet for 56 days
||Body weight measured twice weekly
||Blood and tissue collected
||Heparinized microfuge tubes, dissecting kit
||Selenoenzyme activity analyzed
||Enzyme activity (Gpx1, Gpx3, Gpx4, Txnrd)
||RNA isolated and analyzed (qPCR)
||Relative mRNA abundance of selenoprotein transcripts
- CO2 chamber
- Heparinized microfuge tubes
- LightCycler 480 (Roche Life Science)
- Selenium as Na2SeO3
- Saline phosphate buffer (76 mM NaCl, 50 mM sodium phosphate, pH 7.4)
- Hydrogen peroxide
- Phosphatidylcholine hydroperoxide
- 5,5'-dithiobis (2-nitrobenzoic acid (Sigma #D8130, St. Louis MO))
- TRIzol Reagent (Invitrogen #15596-026)
- KAPA SYBR FAST qPCR Kit (KAPA Biosystems #KK4611)
- Primers (see Sunde, 2018)
Procedure: Diets and body weight
- The basal Se-deficient diet is rodent torula-yeast diet containing 0.005 µg Se/diet, supplemented with 100 mg/kg of all-rac-alpha-tocopherol acetate to ensure prevention of necrosis, and supplemented with 0.4% L-methionine to ensure adequate growth.
- Mice are fed the basal diet supplemented with either 0 or 0.2 µg Se/g diet to provide Se-deficient or Se-sufficient diets for 56 days.
- n=3 per diet for C57BL/6J mice; n = 1 per diet for all other strains.
- Body weights are assessed twice a week (there was no difference in bw between Se-deficient and Se-sufficient mice; therefore, data are pooled across diets and presented as Se-deficient mice).
Procedure: Blood and tissue collection
- Mice are euthanized after 56 days on the diets by terminal CO2 overexposure.
- Blood is collected in heparinized microfuge tubes.
- Blood is centrifuged (1500 xg, 15 min, 5°C) to separate plasma from red cells; RBC are resuspended once, centrifuged, and then reconstituted to the original volume using saline phosphate buffer.
- Livers are removed and immediately frozen at -80°C until analysis.
Procedure: Enzyme activity analysis
- Protein concentration is determined by the method of Lowry.
- Glutathione peroxidase: Gpx1 and Gpx3 activities are assayed with 120 µmol/L H2O2.
- Glutathione peroxidase: Gpx4 activity is assayed with 78 µmol/L phosphatidylcholine hydroperoxide.
- Thioredoxin reductase: Txnrd activity is assayed using 5.3 mM 5,5'-dithiobis (2-nitrobenzoic acid).
Procedure: RNA isolation and qPCR analysis
- Total liver RNA is isolated using TRIzol Reagent and cDNA libraries are prepared.
- Relative mRNA abundance is determined in duplicate by quantitative real-time PCR.
- The mouse gene-specific primer sets for 18 selenoproteins are those designed to amplify ~150 base segments and to span an intron-exon splice junction when possible.
- The 14 µL reactions contain 6 µL cDNA arising from 12 ng of total RNA, 6 µL 2X KAPA SYBR FAST qPCR kit reagent, and 1 µL each of 10 µM forward and reverse primers (see Sunde, 2018).
- Reactions are followed in a LightCycler 480.
- Melting curves are generated to confirm the presence of one specific product and standard curves are run for each primer set/tissue combination.
- The second derivative max program is used to determine amplification efficiency following the manufacturer's protocol.
- mRNA relative abundance is calculated, accounting for gene-specific efficiencies, normalized to the mean of beta-actin (Actb) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression, and expressed as a percentage of the Se-sufficient level.
- To compare transcript expression of different selenoproteins, relative abundance is normalized for basepair length of the amplified fragment.
- Bi-weekly body weight *
- Selenoprotein mRNA abundance relative to C57BL/6J Se+
- Selenoprotein enzyme activity
* Weekly body weights were accessioned by MPD; all body weight data are available in the Sunde1 download file.