strain: Fischer 344 Sex: male age: ~2-3 months tissue: lung
Treatment protocol
Briefly, rats in the heat-stress arm of the experiment were placed in a floor-standing incubator (Thermo Scientific, Ashville, NC) set at room temperature (RT, 22 °C) 24 hours prior to initiation of heat stress experiments. Rats in the control arm (n=18) were introduced to the incubator environment briefly at ambient temperature until Tc stabilized at ≤ 37.3°C, then weighed and returned to their original cages. Rats in the heat-stress arm were heated in the incubator at 37.0 ± 0.2°C until reaching a Tc of 41.8°C (Tc,Max). Six heat-stressed rats were weighed at Tc,Max and euthanized. The remaining rats were placed in a new cage at ambient housing temperature (22.0 ± 0.2°C) and euthanized at 24 hours (n=6) or 48 hours (n=6). Six time-matched controls were euthanized at times corresponding to Tc,Max, 24 hours, and 48 hours in the heat stress arm. Control and experimental animals were provided food and water ad libitum throughout recovery.
Growth protocol
Briefly, rats were housed under standard laboratory conditions (22°C, 12:12 hours light:dark cycle, lights on at 6:00AM) in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. Chow (Harlan Teklad, LM-485; Madison, WI) and water were provided ad libitum. Rats were implanted with TL11M2-C50-PXT PhysioTel® Multiplus Transmitters (Data Sciences International, St. Paul, MN) to measure core temperature (Tc; ±0.25°C), heart rate (HR; bpm), and mean arterial pressure (MAP; ±3 mmHg).
Extracted molecule
total RNA
Extraction protocol
Heart tissues were pulverized under liquid nitrogen using a SPEX 6750 freezer mill (SPEX Sample Prep, Metuchen, NJ). Total RNA was isolated from the pulverized heart and other tissues (liver, kidney and lung) using Trizol (Invitrogen, Carlsbad, CA) followed by column purification with RNeasy Mini kits (Qiagen, GmbH, Germany) to remove residual salt and organic solvents. Total RNA quality and quantity were evaluated using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA) and verified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE).
Label
biotin
Label protocol
An initial input of 100 ng total RNA per sample was synthesized to cRNA using the Ambion Whole Transcript (WT) Expression Kit (Ambion, #4411974) according to manufacturer’s recommendations. Then 10 µg cRNA was used as a template to synthesize cDNA which were fragmented and labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix, #901524).
Hybridization protocol
Fragmented, labeled cDNA were hybridized to Affymetrix RatGene 1.1 ST array plates on an Affymetrix GeneTitan following manufacturer’s instructions using the GeneTitan Hybridization, Wash and Stain Kit for WT array plates (Affymetrix, #901622).
Scan protocol
Array plates were scanned immediately after hybridization, wash and staining by the Affymetrix GeneTitan using default settings.
Description
LU03C_12-2034
Data processing
Microarray data was processed for background adjustment, normalization, and summarization using the interPLIER gene level method (background = PM-GCBG, normalization method = Sketch-Quantile) in Affymetrix Expression Console software (Version 1.1).
