Biopsy of vastus lateralis of a healthy control, with dietary control. No formal exercise training (<2h/wk)
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was extracted from 20-30mg of muscle. Briefly, samples were homogenized in 1 ml of Trizol reagent (Invitrogen Life Technologies, Australia), and incubated at room temperature for 5 min. After addition of chloroform (200 µl), samples were incubated for 3 min at room temperature, centrifuged for 15 min (12 000 g, 4C) and the aqueous phase, containing the RNA, was precipitated with an equal volume of 100% ethanol. Total RNA was further purified through a Qiagen RNeasy Micro-column (Qiagen, Germany) according to manufacturer’s protocol. RNA was resuspended in 10µL RNase-free water. Samples were amplified using the MessageAmp™ aRNA Kit (Ambion; Austin, Texas, USA) according to the manufactures instructions. Briefly, 0.5µg of total RNA was reverse transcribed to form first strand of cDNA, followed immediately by the formation of the second strand of cDNA, which is then purified. A 12 h in vitro transcription reaction was used to synthesize amplified RNA (aRNA), which was then purified and resuspended in 100µL of RNAse free water. aRNA was then ethanol precipitated by storing overnight at -20C in two volumes of 100% ethanol and 0.1 volume of 3M sodium acetate. aRNA was subsequently centrifuged at 12 000 g for 40 min at 4C, the pellet washed twice with 70% ethanol, then the aRNA resuspended in RNAse-free water to a final concentration of ~5mg/ml.
Label
Cy5
Label protocol
RNA were prepared for labelling using the Superscript™ III indirect labelling system (Invitrogen Life Technologies, Australia) where the aRNA was mixed with 1µl spike RNA, 2µl anchored oligo (dT)20 primer (2.5mg/ml) in a total volume of 18 µL and incubated for 5 min at 70 oC. Next, 6 µl of 5X first strand buffer, 1.5 µL 0.1M dithiothreitol (DTT), 1.5µL of dNTP mix with amino-modified nucleotides, 1µL RNAseOUT ™ (40U/µL) and 2µl of Superscript™ III reverse transcriptase (400U/µL) were incubated for 2 h at 46oC. The aRNA template then removed by hydrolysis with 1N NaOH at 70oC for 10 min and neutralized with 0.3 N acetic acid. The cDNA was purified using Qiagen PCR purification columns (Qiagen,Germany), according to the manufacturer’s instructions. Sample cDNAs were labelled with Cy5 (CyDye Post-labeling reactive Dye Pack Amersham Biosciences #RPN 5661) on the column for 1 hr in the dark. Samples were washed with PE buffer, eluted with 80µl of H2O. This eluate is mixed with 400µl of PB buffer.
Purchase in purified form in solution. This RNA was amplified using the MessageAmp™ aRNA Kit (Ambion; Austin, Texas, USA) according to the manufactures instructions. Briefly, 0.5µg of total RNA was reverse transcribed to form first strand of cDNA, followed immediately by the formation of the second strand of cDNA, which is then purified. A 12 h in vitro transcription reaction was used to synthesize amplified RNA (aRNA), which was then purified and resuspended in 100µL of RNAse free water. aRNA was then ethanol precipitated by storing overnight at -20C in two volumes of 100% ethanol and 0.1 volume of 3M sodium acetate. aRNA was subsequently centrifuged at 12 000 g for 40 min at 4C, the pellet washed twice with 70% ethanol, then the aRNA resuspended in RNAse-free water to a final concentration of ~5mg/ml.
Label
Cy3
Label protocol
Universal RNA was prepared for labelling using the Superscript™ III indirect labelling system (Invitrogen Life Technologies, Australia) where the aRNA was mixed with 1µl spike RNA, 2µl anchored oligo (dT)20 primer (2.5mg/ml) in a total volume of 18 µL and incubated for 5 min at 70 oC. Next, 6 µl of 5X first strand buffer, 1.5 µL 0.1M dithiothreitol (DTT), 1.5µL of dNTP mix with amino-modified nucleotides, 1µL RNAseOUT ™ (40U/µL) and 2µl of Superscript™ III reverse transcriptase (400U/µL) were incubated for 2 h at 46oC. The aRNA template then removed by hydrolysis with 1N NaOH at 70oC for 10 min and neutralized with 0.3 N acetic acid. The cDNA was purified using Qiagen PCR purification columns (Qiagen,Germany), according to the manufacturer’s instructions. Sample cDNAs were labelled with Cy3 (CyDye Post-labeling reactive Dye Pack Amersham Biosciences #RPN 5661) on the column for 1 hr in the dark. Samples were washed with PE buffer, eluted with 80µl of H2O. This eluate is mixed with 400µl of PB buffer.
Hybridization protocol
Both labelled sample and reference RNA were added to a new column, Cy3 (reference) then Cy5 (sample). This column was washed with PE buffer, and the pooled DNA eluted with 60µL H2O. The solution was dried in a heated vacuum, then the pellet resuspended and mixed in 16.2 µL of H2O, 30 µL of fresh 2x Hybridization buffer (500µL formamide, 500 µL 10 X standard saline citrate [SSC] and 20µL of 10% sodium dodecyl sulphate [SDS]), 5 µL of Cot1 DNA (Invitrogen), 3.8 µL PolyA (10mg/mL; Sigma) and 5 µL Salmon Sperm DNA (10mg/mL; Invitrogen). The samples were heated to 100oC for 2min, and cooled to room temperature, before the 60µL of solution were applied to the glass micro-array slide and cover slip via capillary action.
Scan protocol
The micro-array slides were scanned using a Molecular Dynamics GenePix 4000B UV laser micro-array scanner (Amersham Pharmacia Biotech, Piscataway, NJ) interfaced with a personal computer utilising GenePix v 5.2 (Amersham Pharmacia Biotech, Piscataway, NJ)
Description
No additional information required
Data processing
Data were imported into Gene Spring (Silicon Genetics, USA) version 7.1 and unless otherwise indicated statistical analysis was performed using default settings in this software. Per chip-intensity-dependent normalization (LOWESS) was carried out for each array and a group of 5962 genes were identified that satisfied the criteria of a mean signal intensity of at least 150 in at least 50% of the hybridizations. Parametric analysis of variance (Welsh ANOVA) was performed on log-transformed ratios of the normalized data for these 5962 genes with Benjamini–Hochberg false discovery rate correction at P 0.05, to identify differentially expressed genes across three populations of athletes.
