Gender: Male Age (years): 56 Height (m): 1.79 Weight (kg): 92.8 BMI: 29 VO2 max: 32.6 IL-6 (pg/ml): 2.92
Samples were collected at the School for Health, University of Bath, Bath, UK
Individuals completed a 24 week exercise training programme. For the first two weeks, individuals completed three exercise sessions per week and from week three onwards, this increased to four sessions per week. Intensity and duration were increased in a progressive manner across the 24 week programme. Subjects completed their exercise sessions in the Fitness Suite at the University of Bath. Their attendance, exercise duration and heart rate for each exercise were recorded using an electronic web-based monitoring system (Fitronics, Bath, UK). Once every fortnight, volunteers reported to the laboratory to complete one of four weekly exercise sessions so that intensity and heart rate response could be monitored and the prescription altered accordingly. For each exercise session, two-thirds of the total duration was spent on a treadmill with the remainder being selected from a choice of a semi-recumbent cycle ergometer, upright cycle ergometer or cross-trainer. Adherence to exercise training during the entire six-month period was calculated for each individual by dividing the total number of sessions performed during the exercise programme by the number of sessions prescribed for the training period x 100%. Venous blood samples were collected after subjects had been in a supine position for 15 min. Prior to blood sample collection all volunteers were asked to refrain from strenuous activity and consuming alcohol for 24 h. Additionally, individuals were asked to refrain from taking medication (e.g. aspirin, paracetamol, ibuprofen) 48 h prior to the blood sample. Sixty ml of whole blood was collected by venepuncture in the ante-cubital vein. Ten ml was dispensed into tubes and left to clot at room temperature for 15 min in order to collect serum with the remainder dispensed into EDTA-coated tubes (Sarstedt Ltd., Leicester, UK). Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated blood using a one-step centrifugation technique (Lymphoprep, Nycomed, Norway). Harvested cells were washed twice in 20 ml PBS and then counted using an automated haematology analyser (SF-3000, Sysmex UK Ltd., UK). PBMCs were lysed in Trizol (Invitrogen, Paisley, UK), with the volume added dependent on cell concentration (1 ml per 5 106 cells), and then frozen at -70°C until subsequent analysis. Serum was analysed for interleukin-6 (IL-6) using a commercially available solid phase high-sensitivity ELISA (Quantikine, R and D Systems Inc., Abingdon, UK).
Twenty sedentary male volunteers were recruited from the local community (primarily though newspaper advertisement) for this 24 week study. Subjects were interviewed by telephone and individuals who had known disease (e.g. heart disease, diabetes, arthritis) or who engaged in structured physical activity on more than two occasions per week were excluded. In addition, volunteers who smoked, had a BMI ≥ 35, or took regular medication were excluded from taking part. Participants who passed the initial screening were invited to attend an information session during which they completed a health questionnaire and were fitted with a physical activity monitor (Actiheart, Cambridge Neurotech Ltd., UK). Subjects were blinded to the purpose of this measurement and the data were used to objectively establish that they were inactive and not meeting current physical activity guidelines. Each volunteer provided written informed consent prior to participating in this investigation which was approved by the local ethics committee. Maximal oxygen uptake (V.O2max) of each subject was determined using a submaximal speed test followed by an incremental incline test on a treadmill ergometer (Woodway, ELG 70 Weiss, Germany). The submaximal running protocol consisted of four steady-state exercise stages, each lasting four min, with speed increasing by 0.8 km.h-1 with each stage. One-minute expired air samples were collected at the end of each stage of exercise using Douglas bags. Measures of heart rate (HR) (Vantage NV, Polar Electro OY, Finland) and rate of perceived exertion (RPE) using a 6-20 scale were taken during the gas collection. Following adequate recovery subjects completed the incremental incline test, consisting of three-minute exercise stages with the incline increasing by 3% at the end of each stage. The test was performed to volitional fatigue to measure V.O2max and maximal heart rate. Expired gas samples were collected in the final min of each stage and during the final minute of exercise along with HR and RPE. Participants were required to meet two out of four standard criteria for having achieved V.O2max: heart rate ≥ than age-predicted maximum heart rate, respiratory exchange ratio ≥ 1.10, rating of perceived exertion ≥19 or reaching a plateau in V.O2.
Total RNA was isolated using the Qiagen RNeasy kit Qiagen. The integrity of the RNA was confirmed with analysis by the Agilent 2100 bioanalyser using the RNA 6000 LabChip kit.
400 ng RNA and 4 µl (1 : 5000 dilution) Agilent One-Color RNA Spike-In RNA were labelled with the Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color according to Manufacture’s instructions as follows: 1.2 µl T7 Promoter Primer was added to 400 ng RNA and 4 µl spike in control and denatured at 65 ºC. First Strand Buffer, DTT, dNTP MMLV and RNaseOut was added. The cDNA was synthesised during the following incubation step (2h at 40 ºC). After 10 min denaturation at 65 ºC and the addition of Cy-labelled CTP, Transcription Buffer, DTT, NTP, PEG, RNaseOUT, Inorganic Phosphatase, and T7 RNA Polymerase the synthesis of the fluorescent labelled cRNA was performed during the second incubation step (2 h at 40 ºC). The labelled cRNA was purified with the Qiagen RNeasy Mini Kit according to Manufacturer’s protocol.
The Agilent Hybridisation Kit (catalogue number 5188-5242) was used in conjunction with Agilent Mouse Oligo Arrays (catalogue number G4122F). 2μg of the labelled sample RNA were used for hybridisation according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol The hybridisation was performed for 17 h at 65 ºC at 10 rpm. Slides were them washed for 1 min at 22 ºC in Wash Solution 1 (catalogue number 5188-5325) and 1 min at 22 ºC in Wash Solution 2, pre-warmed to 37 ºC (catalogue number 5188-5326). Slides were incubated for 30s in Agilent Stabilisation and Drying Solution (catalogue number 5185-5979).
Microarrays were scanned using the Agilent Technologies Scanner G2505B US45102871 (ChipScan software version A.7.0.1), scan region 61 X 21.6mm, scan resolution 5 micron single pass, extended dynamic range selected (XDR Hi 100%; XDR Lo 10%).
This study looks at the effects of physical activity on mRNA expression profiles of the Peripheral Blood Mononuclear cells at several time points and after several weeks of physical activity.
Data was extracted from the scanned image using the Agilent Feature Extraction Software version. 188.8.131.52 (protocol GE1-v5_91_0806). The VALUE column came from the gProcessedSignal column of the result table.