Suppliers:
Amersham Biosciences Uppsala, Sweden
Becton, Dickinson and Company (BD) Ireland, UK
Bio Basic Inc. Ontario, Canada
Biometra (an analytik Jena company) Göttingen, Germany
Bio-Rad California, USA
Biotek Instruments. Inc. Vermont, USA
Boeco Hamburg, Germany
Damad, The National Medical Products co. Ltd Riyadh, Saudi Arabia
Eppendorf Hamburg, Germany
Eurofins MWG Operon LLC Alabama, USA
GE healthcare Buckinghamshire, UK
GFL Gesellschaft for Labortechnik Burgwedel, Germany
Health o meter® Professional Scales Florida, USA
Labcaire New York, USA
Lab-Line Kerala, India
LG Electronics Seoul, South Korea
New England Biolabs Massachusetts, USA
Nichipet Texas, USA
Panreac Darmstadt, Germany
Promega Wisconsin, USA
Pyrex California, USA
Qiagen Boston, USA
Sanyo Biomedical Osaka prefecture, Japan
Sarstedt Nümbrecht, Germany
Shelton Scientific Iowa, USA
Siemens Munich, Germany
Torbal New York, USA
Ufc Biotechnology New York, USA
Western Chemical, Inc Washington, USA
Materials
This section highlights the specific materials used throughout the course of the research project.
Blood collection
Alcohol swabs Damad
Cotton pads BD
EDTA and potassium oxalate vacutainer tubes BD
Sterile needles BD
Tourniquet BD
DNA extraction
DNA extraction kit GE healthcare
1.5 mL sterile microcentrifuge tubes Eppendorf
100, 200, 1000 μL Filtered tips Eppendorf
Sterile transfer pipettes Sarstedt
DNase-free water Qiagen
Genotyping
0.1-10 µL, 200 µL Filtered tips Eppendorf
Primers Eurofins MWG Operon
0.2 mL and 0.5 mL sterile PCR tubes Eppendorf
2x Master Mix Promega
DNase-free water Qiagen
Agarose NA Amersham Biosciences
40X TAE buffer Ufc Biotechnology
Blue/orange 6X loading dye Promega
25 and 100 bp DNA step ladder Promega
Ethidium Bromide Bio Basic Inc.
Glassware Pyrex
Absolute Ethanol Panreac
Equipment
A full set of adjustable micropipettes Eppendorf
Sensitive balance Torbal
Biomedical Freezer Sanyo
Dimension RxL Max clinical chemistry system Siemens
Electrophoresis parts Biometra
Epoc spectrophotometer Bio Tek instruments Inc
Heat-block Eppendorf
Gel imaging system Bio-Rad
Thermal cycler (Mastercycler) Eppendorf
Microcentrifuge Eppendorf
Microwave LG electronics
PCR Workstation (class I hood) Labcaire
Power supply Shelton Scientific
Refrigerator Lab-Line
Sample mixer Sarstedt
Scale Health o meter
Thermomixer Eppendorf
Vortex mixer Boeco
Waterbath GFL Gesellschaft
Methods
This section describes the general methods employed throughout the course of this research project.
The participants were recruited from the out-patient blood collection area at the King Fahad Hospital of the University (KFHU), University of Dammam, Al-Khobar, Eastern province, Saudi Arabia.
Selection of participants and study design
The study was designed as a case-control investigation. It was focused on Saudi Arabian aged between 35 to 70 years, with or without Type-2 diabetes mellitus (T2DM). A total of 152 patients from both sexes with an average age of 47.8 ±8.9 were screened. They were divided into two groups as follows:
a) The control group (84 subjects): healthy non-diabetic subjects
b) Type-2 diabetes mellitus (T2DM) group (68 subjects): participants who were previously diagnosed with T2DM.
Pre-investigation preparation of patients
The individuals recruited for the study were instructed to fast for at least 9 hours before the collection of the blood samples.
Informed consent and ethical approval
Informed consent was collected from all participants involved (IRB-PGS-2015-03-167) in order to obtain an institutional local ethical approval from the University of Dammam, Saudi Arabia.
Data collection
A sample collection form was designed to retrieve demographic data from each participant and included a request for information related to factors such as the current age, gender, age at diagnosis time and family history of diabetes. Height, weight, and waist circumference were measured at the time of collection. The body mass index (BMI) was calculated from the weight (kilograms: kg) and height (meters: m) using the following formula:
BMI = (weight (kg)/height m2)
Blood sample collection
Venous blood samples were retrieved from each subject and collected into two 4.0 mL vacutainer tubes containing EDTA (lavender top) and potassium oxalate (gray top) anticoagulants. The sampling was performed aseptically using a phlebotomy technique. The tube containing the mixture of blood and EDTA was used for DNA extraction analysis. The potassium oxalate tubes were sent to the clinical chemistry laboratory at King Fahad Hospital of the University for fasting plasma glucose measurement.
Measurement of the plasma fasting glucose
Upon arrivals of the samples in the laboratory, centrifugation at 2,400 x g for 8 minutes was performed at room temperature to separate the plasma from other cellular components. The plasma extract from each sample was then used to measure the glucose content by spectrophotometry using the hexokinase (HK)/glucose-6-phosphate dehydrogenase (G-6-PDH) method and the Siemens Dimension RxL Max clinical chemistry system (Siemens, Munich, Germany). First, glucose is phosphorylated by the HK enzyme in the presence of adenosine-5’-triphosphate (ATP) and magnesium ions to generate glucose-6-phosphate (G-6-P). Further, the G-6-P is oxidized by G-6-PDH in the presence of nicotinamide adenine dinucleotide (NAD) leading to the production of 6-phosphogluconate and NADH. The absorbance of NADH, which is directly proportional to the concentration of glucose present in the sample is then determined using a bichromatic (340 and 383 nm) endpoint technique. The reactions depicted below summarize the chemical process previously described.
Glucose + ATP ----HK, Mg2+---> Glucose-6-phosphate +ADP
Glucose-6-phosphate + NAD+ ----G-6-PDH---> 6-phosphogluconate + NADH + H+
Genotyping
The UCP-2 45bp insertion/deletion polymorphism was detected using the standard polymerase chain reaction described in the following sections.
DNA extraction
The genomic DNA was extracted from 200 μl of whole blood using a DNA extraction kit (GE Healthcare Life Sciences Ltd, Buckinghamshire, UK) and according to the following steps. The blood samples were mixed on a roller mixer for 20 minutes and 200 µL transferred into a 1.5 mL microcentrifuge tube followed by the addition of 20 µl of the proteinase K enzyme (20 mg/dL). For each patient, two tubes labeled with the patients’ code numbers were prepared. To lyse the cells, 400 µl of lysing buffer (containing chaotropic salt: 10 mM KHCO3, 155 mM NH4Cl, and 0.1 mM EDTA at pH 8) were added, followed by 15 seconds of vortexing. Further, the tubes were incubated for 10 minutes at room temperature with intermittent vortexing then pulse-spun in the microcentrifuge for 5 seconds at 11,000 × g. The cell lysate was transferred into a pre-labeled minicolumn for DNA binding and spun for 1 minute. The flow-through was removed leaving the DNA bound to the silica gel membrane. The DNA was then washed twice by passing 500 µl of a washing buffer (containing 70% ethanol) through the column to remove residual salts and other contaminants. Finally, the DNA was eluted into a clean pre-labeled microcentrifuge tube with 200 µl of pre-heated (70C) elution buffer.
Measurement of the DNA concentration
The concentration of the extracted genomic DNA was determined using the Epoc spectrophotometer (Biotek Instruments. Inc., Vermont, USA). Samples were read in duplicate along with a suitable blank solution. All readings were saved into an Excel worksheet.
Polymerase chain reaction (PCR)
The DNA locus in the UCP2 gene flanking the polymorphism was first amplified by PCR. The PCR was performed using a 2 x master mix (Promega; Wisconsin, USA), a set of primers and the DNA samples as a template. The forward primer; (5' CAGTGAGGGAAGTGGGAGG3') and the reverse primer; (5'GGGGCAGGACGAAGATTC3') were synthesized by Eurofins MWG Operon LLC (Alabama, USA). Primers were received as lyophilized powder. They were dissolved in sterile distilled water to make the stock solution (100pmol/µL) and stored at -20C until required. The solution of each working primers mix used for the PCR was prepared from the stock solution by making a 1:10 dilution (10pmol/µL) in sterile distilled water. The PCR reaction mixture was set up in 0.2 ml PCR tubes to a final volume of 12.5 µl as described below.
The PCR mixture was transferred into a PCR thermal cycler (Eppendorf, Hamburg, Germany). The standard PCR parameters were as follows:
As a negative control, a separate PCR reaction containing all components except the DNA template was made in every PCR experiment.
Agarose gel electrophoresis
Agarose gel electrophoresis is a preferred method to separate a mixed population of DNA or RNA by their size. During the course of this thesis, agarose gel electrophoresis was used to check the size of the amplified PCR products or enzyme-digested DNA fragments. The percentage (w/v) of agarose gel was dependent on the size of DNA fragments to be analyzed. For the detection of the PCR products, 2% agarose gel was used to visualize DNA fragments.
In preparation for the loading process, the PCR products were individually pre-mixed with an appropriate amount of the 6x loading dye (Promega, Wisconsin, USA) to reach a final dilution of x1 (e.g. 2 µl of the leading dye with 10 µl of the DNA sample). DNA ladder (100bp) of known fragment sizes was run on the same gel to estimate the DNA sizes. It was also pre-mixed with a 6x loading dye as described above. The samples (10 µl) were loaded into the gel wells starting with the DNA ladder in the first well followed by the DNA from the blood samples in the remaining wells. The current was set at 300 volts for 30 minutes. After the running time, the DNA bands were visualized under UV light and imaged using the Bio-Rad gel imaging and documentation system (Bio-Rad, California, USA).
Interpretation of the agarose gels
The positive amplification of the UCP-2 45bp insertion/deletion polymorphism was screened on the gels. This amplification is expressed by the presence of fragments exhibiting sizes of about 412 bp and 457 bp for deletion and insertion respectively. The results from the different samples were compared and grouped according to the DNA profile obtained.
Statistical analysis
