We followed previously published protocols (Feldhoff et al., J Biol Chem 252:3611-6; Tolman et al., J Biol Chem 248:4552-60) for single-pass perfusion. For perfusion of Perk +/+ and -/- mice, the final CaCl2 concentration was 0.1 mM in perfusion buffer. 2,5-di-tert-butylhydroquinone (tBuHQ; Sigma), a selective inhibitor of ATP-dependent microsomal Ca2+ sequestration, was used to induce ER stress. Discontinuous, 9-step 20% (10 mM HEPES, pH 7.4; 250 mM KCl; 5 mM MgCl2; 0.5 mM EDTA; 20% w/w sucrose) to 47% (10 mM HEPES, pH 7.4; 250 mM KCl; 5 mM MgCl2; 0.5 mM EDTA; 47% w/w sucrose) sucrose density gradients were used to separate mRNAs based on the number of bound ribosomes. One mL of detergent-containing supernatant (from perfused liver homogenate) was loaded onto the 20-47% sucrose gradients and centrifuged at 28,000 rpm (Beckmann SW-28), 4°C, for 3 h 38 min for polysome profiles or 22,500 rpm (Beckmann JS-24.38), 4°C, for 19 h 9 min for subunit profiles. Gradient profiles were visualized using an ISCO UV detection unit with a 254 nm filter and fractionated using an ISCO gradient pump (Teledyne Isco, Inc., Lincoln, NE).
Growth protocol
All animals received water and food (Harkland, TX) ad libitum and were maintained according to the Institutional Animal Care and Use Committee-approved protocol at The Pennsylvania State University College of Medicine. Alb/Cre Perk-/- and wild-type littermate mice, 3-6 months old, were generated as previously described (Zhang et al., Mol Cell Biol 22:3864-74)
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from one mL of detergent-containing supernatant or from the sucrose density gradient fractions using acid phenol:chloroform (Ambion, Inc., Austin, TX). Per mL of sample, 40 μL of 0.5 M EDTA, 25 μL of 20% SDS and one volume of acid phenol:chloroform were added. After a 30 min centrifugation at 3, 000 x g (Beckmann GS-6KR), 4°C, the top clear layer was removed, and RNA extracted a second time with the addition of one volume acid phenol:chloroform. Precipitation of RNA was done overnight in 2.5 volumes of 100% ethanol and 1/10 volume of 5 M NH4OAc (Ambion). RNA was pelleted, washed twice with 75% ethanol and resuspended in RNA storage solution plus anti-RNAse (Ambion). The concentration and purity of RNA samples were measured by the Agilent 2100 Bioanalyzer (Robert Brucklacher, Functional Genomics Core Facility, PSU-COM) prior to hybridization array and QRT-PCR applications.
Label
biotin
Label protocol
RNA from two perfused liver samples per condition (8 conditions: unfractionated and polysomal fractions of treated or control wild-type or knockout) were pooled to minimize biological variations. Twelve μg of total RNA were used in the initial cDNA conversion step. All steps including double-strand cDNA synthesis, in vitro labeling and cRNA fragmentation were done using the Affymetrix One-Cycle Target Labeling and Control Reagents kit and protocol.
Hybridization protocol
15 ug of fragmented cRNA was used to prepare 300 ul of hybridization cocktail as per the manufacturer's instructions. 200 ul of that hybridization cocktail was hybridized to Mouse Genome 430_2.0 Arrays for 16 hours at 45C. Arrays were washed and stained using a GeneChip Fluidics Station 400 as per the manufacturer's instructions.
Scan protocol
Arrays were scanned using the Affymetrix GCS 3000 scanner.
Description
Unfractionated sample
Data processing
Affymetrix CHP files containing qualitative and quantitative values of each probe set on the arrays were imported into GeneSpring (Agilent Technologies, Inc., Palo Alto, CA) and sorted for raw expression values > 80 in at least 1 of 16 conditions as an initial filter to eliminate background noise. The resulting lists were imported into Excel and analyzed using the Z-ratio method (Cheadle et al., J Mol Diagn 5:73-81; Mazan-Mamczarz et al., Biotechniques 39:61-7). First, the data were transformed using log10. The transformed values were used in the following calculations: Zscore = (intensityG – mean intensityG1…Gn)/SDG1…Gn, where G = any gene on hybridization array, G1…Gn = aggregate measure of all genes. Zratios = [(ZscoreG1ave)Exp – (ZscoreG1ave)Con]/SD of Zscore differencesG1…Gn, where G1 = average Z score for any particular gene being tested under multiple experimental conditions (experimental vs. control), G1…Gn = aggregate measure of all of the genes. Z ratio data is provided as a supplementary file on the Series record.
