Initially, cells were incubated at 37ºC with 5% CO2 to stabilize the culture. Subsequently, the plate marked as treated was exposed to 42ºC for one hour to simulate heat stress condition. After 1h, the cells were allowed to recover at 37ºC, 5% CO2 and harvested by trypsinization at different time points (30m, 2h, 4h, 8h, 12h, 16h, and 24h).
Growth protocol
The primary MECs derived from buffalo mammary gland were cultured using DMEM/F12 and supplements. After 10th passages, 80% confluent buffalo MECs were distributed in collagen treated 12-wellplates in two sets with one plate assigned as control (kept at 37ºC all the time) and other plate as treated (exposed to 42ºC).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from MEC using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. After extraction, RNA was purified using RNeasy Mini kit (Qiagen, Germany) and followed by on-column digestion with the RNase-free DNase (Qiagen, Germany). RNA was quantified using Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies). RNA integrity was confirmed by denaturing agarose gel electrophoresis and experion bioanalyzer (BioRad, USA,).
Label
Cy3
Label protocol
For One-Color Microarray-Based Gene Expression Analysis using Low Input Quick Amp Labelling kit (Agilent Technologies, Santa Clara, CA), the RNA samples (CTR, 30 m, 2h, 4h, 8h, 12h, 16h, 24 h) were labelled with T7 promoter primer (65°C for 10 min followed by incubation in ice for 5 min). cDNA was constructed from labelled RNA samples after adding cDNA master mix (5X First Strand Buffer, 0.1 M DTT, 10 MmdNTP mix and AffinityScriptRNase Block Mix) followed by incubation at 40°C for 2 h, 70°C for 15 min and final incubation on ice for 5 min. The cRNA synthesis and amplification was performed by adding transcription master mix (5X Transcription Buffer, 0.1 M DTT, NTP mix, T7 RNA Polymerase Blend and Cyanine 3-CTP) followed by incubation at 40°C for 2 h. The amplified labelled cRNA was purified (RNeasy mini column kit, Qiagen, Germany), quantified [μgcRNA yield = (Concentration of cRNA) x 30 µL (elution volume)/ 1000] and checked for specific activity [pmol Cy3 per µg cRNA = (Concentration of Cy3/ Concentration of cRNA) x 1000]. All the samples exhibited yield and specific activity values higher than the minimum value of 1.65 and 6, respectively using four pack microarray format.
Hybridization protocol
For hybridization, 1.65 μg of linearly amplified and cyanine 3-labeled cRNA were fragmented using fragmentation mix (10X Blocking Agent and 25X Fragmentation Buffer) and incubated at 60°C for exactly 30 min. The fragmented RNA samples were immediately transferred on ice for one minute and 55μl of 2x GEx Hybridization Buffer HI-RPM was added to stop the fragmentation reaction. The fragmented samples (110μl volume) were loaded to the array placed in slide chamber. The assembled slide chamber placed in rotisserie was put in a hybridization oven set to rotate at 10 rpm and 65°C. The hybridization was allowed for 17 hours. Followed by hybridization, microarray slide was disassembled in GE wash buffer 1 (pre warm overnight at 37°C) containing Triton X-102 (0.005%), washed with fresh GE wash buffer 1 for 1 min followed by second wash with GE Wash Buffer 2 for 5 min at room temperature. The slide was dried and scanned immediately.
Scan protocol
Immediately after washing, the slides were scanned on GenePix-4000B (Molecular Device) microarray scanner using one color scan setting with 5μm resolution at wavelength of 532 nm (Cy3). After scanning, the images were collected as 16 bit images and finally stored as tif image files.
Description
Gene expression of buffalo MEC after 4h min of heat stress BMEC heat stressed 4h_GE1-v5_95_4
Data processing
These files were further subjected to feature extraction using Agilent Feature Extractor Software Version 9.5 (Agilent Technologies) software. The data was normalized to the 75th percentile using GeneSpring GX 11.5 (Agilent Technologies). Genespring GX 12.6 (Agilent technologies) was used to perform the analysis of raw data obtained from feature extraction software.
