Contents & References of Investigating the expression level of liver alpha-amylase gene by Real time PCR method in the liver tissue of normal and obese mice
List:
Abstract: 1
Chapter 1: General 2
1-1-Statement of the problem. 3
1-2- Research objectives. 4
1-3- The main assumptions of the research. 4
1-41- The aspect of newness and innovation in research. 4
Chapter 2: Review of past studies 5
2-1-Amylase 6
2-1-1- History. 6
2-1-2-amylase in human development. 6
2-1-3- Functions of amylase 7
2-1-4- Structure of amylase 9
2-1-5- Measurement of blood level. 9
2-2-alpha-amylase 10
2-2-1-function of alpha-amylase 11
2-2-2-alpha-amylase in human physiology. 12
2-3- Salivary amylase or Ptyalin 12
2-3-1- Genetic variation in salivary amylase. 12
2-4- Pancreatic alpha-amylase 13
2-5- Alpha-amylase in pathology 13
2-5-1- Interpretation of alpha-amylase level 13
2-6- Alpha-amylase limiting buffer 14
2-7- Enzyme activity study 14
2-7-1- Alpha-amylase activity measurement methods before the colorimetric method. 14
2-7-2 - Colorimetric methods: 15
2-8- Domain architecture of alpha-amylase structure (Domain) 15
2-9- Alpha-amylase genes in humans 16
2-10- Alpha-amylase in mice 18
2-10-1-Difference in the end sequence of 5 mRNAs Hepatic alpha-amylase and rat salivary glands in terms of size 19
2-10-2- Studies related to analysis of liver alpha-amylase mRAN sequence. 20
2-10-3-characteristics of hepatic alpha-amylase mRAN. 20
2-11- alpha-amylase inhibitors 21
2-11-1- green tea 22
2-11-2- benefits of green tea 22
2-11-2-1- prevention of blood clot disease 23
2-11-2-2- the effect of green tea on people suffering from tri Glyceride. 24
2-11-2-3-thinning the blood and preventing it from clotting. 24
2-11-2-4-reducing blood pressure and preventing it. 24
2-11-2-5-minimizing the amount of damage to the brain and heart. 25
2-11-2-6-Protection against all types of cancer. 25
2-11-2-7-prevention of type 2 diabetes. 25
2-11-2-8-weight loss. 26
Chapter 3 Materials and Methods 27
3-1- Working methods: 28
3-1-1- Laboratory animal under study: 28
3-1-1-1- The reasons for choosing this animal in brief are: 28
3-1-1-2-Animal keeping conditions: 28
3-1-2- The type of study 29
3-2- The method of implementing the project (fattening mice using a high-fat diet) 29
3-2-1- Preparation of aqueous and alcoholic extract of green tea 30
3-2-2- Separating the liver and drawing blood from the mouse heart. 30
3-3- RNA extraction 34
3-3-1- RNA extraction tools. 34
3-3-2- RNA extraction steps. 34 3-3-2-1- Precipitation of RNA: 35 3-3-2-2- Washing of RNA: 35 3-3-2-3- Drying of RNA sediment: 35 3-3-3 Quantitative and qualitative assessment of extracted RNA: 36 3-3-3-1- Measurement of RNA quantity: 36
3-3-3-2- RNA quality check: 36
3-3-3-3- RNA conservation: 37
3-4- Gene expression check 37
3-4-1- RT-PCR. 37
3-4-2- cDNA replication. 39
3-4-2-1- PCR(Polymerase Chain Reaction) 39
3-4-2-2- Materials and tools needed to make cDNA. 43
3-4-2-3- The method of performing cDNA production test. 44
3-4-2-4- how to perform the RT-PCR reaction to ensure the accuracy of cDNA synthesis. 45
3-4-2-5- Detection of PCR products by electrophoresis 47
3-4-2-6- Devices required for electrophoresis 47
3-4-2-7- Necessary materials for electrophoresis 47
3-4-2-8- How to perform electrophoresis 48
3-4-2-9- Gel detection. 48
3-5- Real-time PCR 49
3-5-1- Use cases of Real-time PCR. 49
3-5-2- Different phases of a real time PCR reaction. 50
3-5-3- Real-time PCR assay methods. 51
3-5-3-1- non-specific assay method with Real-time PCR. 51
3-5-4- Threshold concept and Ct. 52
3-5-5- How to draw Melt Curve Analysis 53
3-5-6- How to do Real time PCR. 55
3-5-6-1- Necessary materials to perform Real time PCR. 55
3-5-6-2-method of performing Real time PCR test. 55
3-5-6-3- Statistical analysis 57
3-6-Measurement of ?-Amylase in serum55
3-5-6-3- Statistical analysis 57
3-6-Measurement of ?-Amylase in mouse serum: 57
3-6-1-Separation of serum from mouse blood. 57
3-6-2- Measurement of ?-Amylase in serum by photometric method. 57
Chapter 4 Results. 60
4-1- Weight gain observations of test group mice 61
4-2- Determining the quality and concentration of extracted RNA 66
4-3- Checking the quality of cDNA synthesized by PCR 67
4-4-Melt Curve Analysis 68
4-4-1- Comparison of liver alpha amylase gene expression in mice Fat and normal. 70
4-4-2-The effect of green tea on liver alpha amylase gene expression in obese mice. 71
4-5- Measurement of alpha-amylase in serum by photometric method 72
Chapter five: Discussion and conclusion 73
5-1- Discussion and conclusion 74
5-2- Suggestions 81
Resources 83
English summary 92
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