Projects
Name
Polymorphism of ferric reductase gene in adolescents girls with anemia
University
Indonesia (CIMSA-SIMKI) - Universitas Sebelas Maret, Surakarta
Domain
Biochemistry
Departement
Biomedical Laboratory, Faculty of Medicine, Universitas Sebelas Maret Jl. Ir. Sutami 36A, Kentingan, Surakarta 57126, Central Java, Indonesia
Head
Dono Indarto, MD, M.Biotech.St., Ph.D
Tutor
Dono Indarto, MD, M.Biotech.St., Ph.D
Languages
English, Indonesian
Duration
4 weeks
Availability
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
Yes Yes Yes No No No No No No No No No
Type of Research Project
- Basic science
What is the background of the project?
Anemia is an iron metabolism disorder that commonly occurs in susceptible age groups such as children, adolescent girls and pregnant women around the world. Iron deficiency is the most important factor that underlies anemia especially in developing countries. Anemia in adolescent girls has wide impacts in their future life. Beside inadequate food intake that contains high iron levels, genetic factor also contributes to the anemia. For example, ferric reductase enzyme plays an central role in converting ferric ion into ferro ion in the present of vitamin C in the small instetine. Polymorphism in the last exon of this gene (Dyctb) enhances ferric reductase acitvity in an in vitro study using Chinese hamster ovary cells.
What is the aim of the project?
To investigate the influence of ferric reductase polymorphism on hemoglobin levels in anemic adolescent girls in Boyolali District and Sukoharjo District who get iron supplementation.
What techniques and methods are used?
1. Collection of blood sample from median cubiti vein of all subjects into vacutainer tubes with Ethylenediaminetetraacetic Acid (EDTA) anti-coagulant 2. Measurement of haemoglobin level using cyanmethemoglobin method • Measure 4 ml of diluent (Drabkin’s solution) with caution into a test tube (T) • Add 0.02 ml of well mixed blood to the diluent and cover • Mix by inversion upside-down several times • Let stand for 10 minutes at room temperature in darkness • Read absorbance of (T) on a spectrophotometer at 540 nm against a blank (Drabkin’s reagent) • Find the concentration of (T) in g/L from a standard curve relating absorbance reading to Hb concentration in g/L 3. Measurement of the plasma ferritin levels using human ferritin Enzyme Linked Immunosorbent Assay (ELISA) kit • As it is necessary to perform the determination in duplicate, prepare two wells for each of the six points of the standard curve (S0-S5), two for each sample, one for Blank • Pipette 20 μL of sample to the sample well • Pipette 20 μL of standard S0-S5 to the standard well • Pipette each 100 μL of conjugate to the standard and sample wells • Incubate at room temperature for 1 hour • Remove the contents from each well • Wash the wells with 300 μL of diluted wash solution • Repeat the washing procedure two times by draining the wash completely • Pipette 100 μL of TMB-Substrate to all wells • Incubate at room temperature for 10 minutes in the dark • Pipette 100 μL of stop solution to all wells • Read the absorbance at 450 nm against blank 4. Isolate the Dyctb gene exon 4 from peripheral blood nuclear cells using Deoxyribose Nucleic Acid (DNA) isolation kit 5. Amplification of the Dyctb exon 4 with Polymerase Chain Reaction (PCR) master mix • Denaturation at 94⁰C for 5 minutes and followed by 35 cycles: denaturation at 94⁰C for 20 seconds, annealing at 56⁰C for 30 seconds, and elongation at 72⁰C for 60 seconds. Final extension is then carried out at 72⁰C for 60 seconds • Denaturation: Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step • Annealing: Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA • Extension: Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing 6. Electrophoresis of amplified products used 2% agarose gel and visualization using cyber green • Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. • The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end. The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result, the molecules are separated by size. 7. All collected data will be analyzed using Chi square and Pearson tests with p values < 0.05 through Statistical Package for Social Sciences (SPSS) 24
What is the role of the student?
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
1. Measurement of haemoglobin level using cyanmethemoglobin method • Measure 4 ml of diluent (Drabkin’s solution) with caution into a test tube (T) • Add 0.02 ml of well mixed blood to the diluent and cover • Mix by inversion upside-down several times • Let stand for 10 minutes at room temperature in darkness • Read absorbance of (T) on a spectrophotometer at 540 nm against a blank (Drabkin’s reagent) • Find the concentration of (T) in g/L from a standard curve relating absorbance reading to Hb concentration in g/L 2. Measurement of the plasma ferritin levels using human ferritin Enzyme Linked Immunosorbent Assay (ELISA) kit • As it is necessary to perform the determination in duplicate, prepare two wells for each of the six points of the standard curve (S0-S5), two for each sample, one for Blank • Pipette 20 μL of sample to the sample well • Pipette 20 μL of standard S0-S5 to the standard well • Pipette each 100 μL of conjugate to the standard and sample wells • Incubate at room temperature for 1 hour • Remove the contents from each well • Wash the wells with 300 μL of diluted wash solution • Repeat the washing procedure two times by draining the wash completely • Pipette 100 μL of TMB-Substrate to all wells • Incubate at room temperature for 10 minutes in the dark • Pipette 100 μL of stop solution to all wells • Read the absorbance at 450 nm against blank 3. Isolation of the Dyctb gene exon 4 from peripheral blood cells using Deoxyribose Nucleic Acid (DNA) isolation kit 4. Amplification of the Dyctb exon 4 with Polymerase Chain Reaction (PCR) master mix • Denaturation at 94⁰C for 5 minutes and followed by 35 cycles: denaturation at 94⁰C for 20 seconds, annealing at 56⁰C for 30 seconds, and elongation at 72⁰C for 60 seconds. Final extension is then carried out at 72⁰C for 60 seconds • Denaturation: Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step • Annealing: Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA • Extension: Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing 5. Electrophoresis of amplified products used 2% agarose gel and visualization using cyber green • Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. • The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end. The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result, the molecules are separated by size. 6. Record and analyze the data to determine percentage of mutated ferireductase gene from total samples using Chi square and Pearson tests with p values < 0.05 through Statistical Package for Social Sciences (SPSS) 24
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Students will have lectures / tutorials on: 1. Anemia in Indonesia (Amelia and Dwi) 2. Central dogma of molecular biology (Dono) 3. Molecular biology techniques (Yuliana) 4. Laboratory works (Research project team)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Laboratory skills (including using micropipettes and handling toxic waste); Subjects passed: basic concept of molecular biology; Previous experience with: laboratory works
Are there any legal limitations in the student’s involvement
Yes
Incoming students will always be in strict supervision of the tutors and/or supervisor. Incoming are not allowed to take the research data outside of Sebelas Maret University premises.
Hours
6
Type of students accepted
This project accepts:
- Medical students
- Graduated students (less than 6 months)
- Pre-Medical students from the American-British system
- Students in biomedical fields
Articles
- Schlottmann; F.; Vera-Aviles; M. and Latunde-Dada; G.O. (2017) Duodenal cytochrome b (Cybrd1) ferric reductase functional studies in cells. Metallomics DOI: 10.1039/c7mt00254h.