Sunde1 project protocol

Selenium regulation of selenoprotein enzyme activity and transcripts in 8 inbred founder strains of the Collaborative Cross   (2018)

Sunde RA




Project protocol - Contents

Workflow and sampling

Step
Procedure
 Equipment
Data collected
1
 Mice fed basal diet supplemented with 0 or 0.2 µg Se/g diet for 56 days -
-
2 Body weight measured twice weekly
 Balance Body weight
3 Mice euthanized CO2 chamber -
4 Blood and tissue collected Heparinized microfuge tubes, dissecting kit -
5 Selenoenzyme activity analyzed Spectrophotometer Enzyme activity (Gpx1, Gpx3, Gpx4, Txnrd)
6 RNA isolated and analyzed (qPCR) PCR machine Relative mRNA abundance of selenoprotein transcripts

Equipment and supplies

  • Balance
  • CO2 chamber
  • Heparinized microfuge tubes
  • Centrifuge
  • LightCycler 480 (Roche Life Science)

Reagents and solutions

  • 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

  1. 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.
  2. 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.
  3. n=3 per diet for C57BL/6J mice; n = 1 per diet for all other strains.
  4. 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

  1. Mice are euthanized after 56 days on the diets by terminal CO2 overexposure.
  2. Blood is collected in heparinized microfuge tubes.
  3. 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.
  4. Livers are removed and immediately frozen at -80°C until analysis.

Procedure: Enzyme activity analysis

  1. Protein concentration is determined by the method of Lowry.
  2. Glutathione peroxidase: Gpx1 and Gpx3 activities are assayed with 120 µmol/L H2O2.
  3. Glutathione peroxidase: Gpx4 activity is assayed with 78 µmol/L phosphatidylcholine hydroperoxide.
  4. Thioredoxin reductase: Txnrd activity is assayed using 5.3 mM 5,5'-dithiobis (2-nitrobenzoic acid).

Procedure: RNA isolation and qPCR analysis

  1. Total liver RNA is isolated using TRIzol Reagent and cDNA libraries are prepared.
  2. Relative mRNA abundance is determined in duplicate by quantitative real-time PCR.
  3. 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.
  4. 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).
  5. Reactions are followed in a LightCycler 480.
  6. Melting curves are generated to confirm the presence of one specific product and standard curves are run for each primer set/tissue combination.
  7. The second derivative max program is used to determine amplification efficiency following the manufacturer's protocol.
  8. 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.
  9. To compare transcript expression of different selenoproteins, relative abundance is normalized for basepair length of the amplified fragment.

Data collected by investigator

  • 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.