Chicken is the most common domesticated animal. Besides being the most important food production animal,chickens can also serve other purposes, such as to provide companionship as pets. Chicken are the most abundant and widely distributed bird in the world. Billions of birds are used in meat and egg production each year. Across the globe, the health of chickens contribute to the wealth of nations. The chicken industry is very important. In United Kingdom (UK) for example, chicken farming remains crucial to the rural economy and it contributes significantly to the balance of trade. At any one time, the UK is home to approximately 29 million egg-laying chickens and more than 100 million chickens bred for meat production. The United States is the world's largest producer of chicken meat. Their expected 2010 production of chicken meat is 16.3 million ton.
Chickens are a major source of energy and protein for humans worldwide. It provides 67.6% of the daily value for protein in 4 ounces. Chicken is also a good source of selenium which is a trace mineral. Selenium is an important component of several major metabolic pathways, including thyroid hormone metabolism, antioxidant defense systems, and immune function.
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Chicken is a very good source of the cancer-protective vitamin,Â niacin. Components of DNA require niacin. A deficiency of niacin, as well as other B-complex vitamins, has been directly linked to genetic (DNA) damage. A research published in the August 2004 issue of the Journal of Neurology, Neurosurgery and Psychiatry indicates regular consumption of niacin-rich foods like chicken provides protection against Alzheimer's disease and age-related cognitive decline.
Diseases that chicken face and overcoming these diseases
Chickens suffer from a variety of diseases including Newcastle disease, mareck's disease and salmonella infections. Newcastle disease, which is an airborne virus , attacks internal organs and causes death in chickens. It can wipe out the entire flock in just a few days. Mareck's disease affects chickens through white blood cells and presents itself as cancerous tumors. Virulent pathogens can also harm chickens. These diseases can cause great losses for farms that are focused on chicken rearing.
In year 2000 a three year programme was started to explore the genetic basis for immunity in chickens and to develop a database of expression profiles for a large set of chicken genes. This project is known as Chicken-Image(Improvement of chicken Immunity and resistance to disease based on Analysis of Gene Expression). Through this project, a range of commercially valuable products to provide the technology for breeding resistant chickens, could be produced.
Antimicrobials have been used extensively in intensive poultry operations to minimise disease, improve growth and feed utilisation. However, antimicrobials in animal diets can cause the development of resistant strains of microbes. This could impact human health directly and carry into meat and bioproducts. Therefore, the European Union has moved towards a complete ban of in-feed antimicrobials for these reasons. Research on alternatives to the present in-feed antimicrobials is being conducted world-wide. In all cases, it will be necessary to minimise disease challenges, strengthen the bird's natural defences (immune response) and to optimise the diet to provide a balance of required nutrients for the chicken's changing needs. All of these may be influenced by using feed additives.
A prebiotic is defined as "a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon" (Gibson.G.R, Roberfroid M.B,1995). Modification by prebiotics of the composition of the colonic microflora leads to the predominance of a few of the potentially health-promoting bacteria, especially, but not exclusively, lactobacilli and bifidobacteria (Gibson.G.R, Roberfroid M.B,1995). Because of the ability of prebiotics to encourage the growth of beneficial bacteria it could be used in the case when there is a need to improve the gut flora. The most common types of prebiotics available in supplements are fructooligosaccharides (FOS), inulin and galactooligosaccharides.
Chicken Immune System
Antigens are microorganisms or toxins that can invade the body and cause diseases. The skin provides a barrier to invading microbes. It is generally penetrable only through cuts or tiny abrasions. The digestive and respiratory tracts, both portals of entry for a number of microbes, also have their own levels of protection. Microbes entering the nose often cause the nasal surfaces to secrete more protective mucus. The stomach contains a strong acid that destroys many pathogens that are swallowed with food. Phagocytosis which is the engulfing and inactivating of antigens as well as lysis of foreign cells can be used to eliminate foreign organisms.
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There are many similarities as well as differences between the general immune mechanisms of mammals and chickens. Birds respond to antigenic stimulation by generating antibodies as well as cellular immunity. There are three principal classes of antibodies in birds. They are IgM, IgG and IgA. Antibody diversity is achieved by gene conversion. T cells are the main effector cells of cellular immunity. The avian T cells differentiate into two distinct pathways which are the alpha/beta and gamma/delta.
Avian T cell diversity is likely generated through combinatorial and junctional mechanisms similar to the mechanisms that operate in mammalian T cell receptors. As in mammals, avian T cells engage in helper and cytotoxic functions that are MHC restricted. The innate effector mechanisms include those mediated by natural killer (NK) cells and antibody dependent cellular cytotoxicity (ADCC). Recently, genes of several avian cytokines have been cloned and expressed. A number of naturally occurring viruses cause immunosuppression in chickens ,therefore, there is much interest in understanding the mechanisms of immunosuppression and developing strategies to enhance immune responsiveness in commercial poultry.
The body contains white blood cells which are made in the bone marrow. These white blood cells are called lymphocytes. The role of lymphocytes is to help protect the body from infection. The three different types of lymphocytes are the B cells, T cells, and natural killer cells. In chickens, lymphocytes are produced in 2 lymphoid organs, the thymus and the bursa of Fabricus. Both B cells and T cells recognize specific antigen targets. B cells are found in the bursa of Fabricus. They work mainly by secreting antibodies into the body's fluids, or humors. They are responsible for the production of immunoglobulins and play a role in the humoral response. These antibodies will interact with circulating antigens such as bacteria and toxic molecules, but they will not be unable to penetrate living cells. T cells are found in the thymus. They interact directly with their targets, attacking body cells that have been taken over by viruses.
Birds have only one variable function gene encoded in its germline DNA. Therefore, the B cells would not be able to produce the different antibodies which would be needed to react against specific diseases. This problem could be solved by gene conversion. Gene conversion is a process whereby the diversity of the antibodies could be rearranged and further immunoglobulin diversity could be provided with the help of hypermutation.
Cytokines are proteins that play many roles in the immune system function. They are usually pleiotropic molecules with diverse and cell type specific activities. Their functions are mediated by binding specific receptors. Cytokines have many activities including, regulating cell activation, hematopoiesis, apoptosis, cell migration, and cell proliferation. Cytokines are involved in almost all aspects of both innate and adaptive immune responses. Our experiment is focused on 3 different cytokines. They are interleukin-1, interleukin-6 and interferon-Î±. These cytokines are expressed in mammalian as well as avian species.
Interleukin-1 (IL-1) consists of two proteins, IL-1 alpha and IL-1 beta. These are the products of distinct genes. Approximately 25% amino acid sequence identity is shown by these proteins which recognize the same cell surface receptors. IL-1 is generally produced in response to inflammation and nervous system stimulation. Due to stimuli produced by inflammatory agents, infections or microbial endotoxins, a vast increase in the production of IL-1 by macrophages and various other cells can be noticed. Osteoblasts, monocytes, macrophages, keratinocytes, Kupffer cells, hepatocytes, thymic and salivary gland epithelium, Schwann cells, fibroblasts and glia (oligodendroglia, astrocytes and microglia) are cells known to produce IL-1.
Interleukin 6 (IL-6) is a pleiotropic Î±-helical cytokine. It plays important roles in acute phase reactions, inflammation, hematopoiesis, bone metabolism, and cancer progression. IL-6 activity is essential for the transition from acute inflammation to either acquired immunity or chronic inflammatory disease. IL-6 is produced by macrophages and T cells. IL-6 can induce pyrogens in muscle and fat tissues, therefore causing the body temperature to increase. They also induce pro-inflammatory response by causing the differentiation of B-cells into immunoglobins secreting cells.
Interferon-alpha (IFN-alpha) is produced by leukocytes. IFN-alpha has both anti-viral and immunomodulatory activities on target cells. Protein kinase and oligoadenylate synthethase are produced when IFN-alpha is stimulated by a viral infection trigger.
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Ginseng has been used as a medicine for over two thousand years. Today, approximately 6 million Americans use it regularly. Asian ginseng is native to China and Korea and has been used in various systems of medicine for many centuries. Chinese ginseng is a key herb in Chinese medicine. Chinese ginseng's Latin name is Panax ginseng. It is a member of the Araliaceae family. The Panax family consists of at least nine species, including P. ginseng, Panax quinquefolium (Xiyangshen, American ginseng), Panax notoginseng (Sanqi) and Panax japonicus (Japanese ginseng).
Ginseng modulates blood pressure, metabolism and immune functions. It has been known to have improved the health of people recovering from illness and boosting the immune system. Ginseng can also increase stamina and improve both mental and physical performance. The National Center for Complementary and Alternative Medicine (NCCAM) has funded research which includes the herb's potential role in treating insulin resistance, cancer, and Alzheimer's disease.
The root of Panax ginseng contains active chemical components called ginsenosides that are thought to be responsible for the herb's medicinal properties. The root is dried and used to make tablets or capsules, extracts, and teas, as well as creams or other preparations for external use. Ginsenosides were isolated in 1963. Until then, the action mechanism of ginseng had not been known. Much effort has since been focused on evaluating the function and elucidating the molecular mechanism of each ginsenoside.
Ginsenosides are triterpene saponins. Most ginsenosides are composed of a dammarane skeleton. Which is a four-ring structure containing 17 carbons with various sugar moieties (e.g. glucose, rhamnose, xylose and arabinose) attached to the C-3 and C-20 positions. Ginsenosides are named as 'Rx'. The 'R' stands for the root and the 'x' describes the chromatographic polarity in an alphabetical order, for example, Ra is the least polar compound and Rb is more polar than Ra.
There are over 30 ginsenosides which have and identified. They are classified in two categories. The first category is the 20(S)-protopanaxadiol (PPD) which contains Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, Rs1. The second category is the 20(S)-protopanaxatriol (PPT) which contains Re, Rf, Rg1, Rg2, Rh1. A carboxyl group is present in the C-6 position of PPDs whereas it is absent in PPTs. Several rare ginsenosides, such as the ocotillol saponin F11 (24-R-pseudoginsenoside) and the pentacyclic oleanane saponin Ro (3,28-O-bisdesmoside) have also been identified.
Different ginsenosides have different functions. Rg1 modulates humoral and cellular immunity. Rh2, Rb1, Rb2 and Rc provide anti-proliferative activity against tumour cells. Rh1, Rh2, Rh3, Rg3 induce tissue differentiation and correct adhesive cellular interactions. Rh enhances DNA-polymerase corrective activity which is important for anti-mutagenity. Rb1, Rg1 and Rg3 inhibit tumour angiogenesis. Rb1, Rb2, Re and Rg1 up-regulate activities of antioxidant enzymes. Rb1 prevents neuronal apoptosis (stimulating Bcl-x(L) expression and down-regulating caspase-3 level.)
Several factors affect the quality and composition of ginsenosides in the ginseng plants. They are the species, age, part of the plant, cultivation method, harvesting season as well as the preservation method. For example, ginsenoside Rf is unique to Asian ginseng while F11 is found exclusively in American ginseng. Therefore, the Rf/F11 ratio can be used as a phytochemical marker to distinguish American ginseng from Asian ginseng. The overall saponin content in ginseng is directly proportional to its age. It reaches a peak level at around 6 years of age.
Two commonly used standardized extracts are G115 from P. ginseng (Pharmaton SA, Switzerland) and NAGE from P. quinquefolius (Canadian Phytopharmaceuticals Corporation, Canada). Studies on these two ginseng extracts using high-performance liquid chromatography (HPLC) found ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1 in both G115 and NAGE, and ginsenoside Rg2 in G115 only. G115 has higher Rg1, but NAGE has higher in Rb1 and Re.
3. MATERIAL AND METHODS
Gallus gallus cell line CRL-12357 was purchased from ATCC, cells were thawed for culturing and maintained.
Exposure of Gallus gallus cells line CRL-12357 to crude ginseng stem and root extracts at different treatment of 0, 24, 48 hours respectively.
Harvesting of treated cells and the no extract controls after each time point 0, 24 and 48 hours.
Total RNA Extraction
Kit: QIAGEN RNeasy Plus Mini Kit
(Catalogue no. 74134)
cDNA synthesis using Invitrogen First-strand synthesis kit.
Data analysis and interpretation of results.
3.1 Gallus gallus T-lymphocyte cell line (ATCC CRL-12357)
The chicken-spleen derived T-lymphocytes cell line (ATCC CRL-12357) was purchased form ATCC (USA). The cells were thawed and grown in RPMI-1640 complete medium (HEPES Modified Sigma Catalogue no. R8005, USA) which contains 2mM-Lglutamine, 10 mM HEPES, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, with 1 mM sodium pyruvate, 20% ( v/v ) foetal calf serum and 1% ( v/v ) penicillin-streptomycin-amphotericin.
3.2 Media preparation
3.2.1 RPMI-1640 basal medium and 4x complete medium
Each bottle of powdered RPMI-1640 (Sigma Catalogue no. R8005) was made into 1 litre of final medium. 3 bottles of powdered medium was dissolved in 2.4 litres of Milli-Q (nano-pure) water in a beaker. Original bottles were rinsed with a small amount of Milli-Q water to remove traces of powder. A Magnetic-stirrer was used to mix the solution till the powder had totally dissolved. Newly constituted medium had a pH of 7.0.
The solution was transferred carefully into a measuring cylinder and brought to the final volume of 3 litres with Milli-Q water and the medium was made sterile by filtration through a 0.2ïm polyethersulfone bottle-top membrane filter (Nalgene, Catalogue no.595-4520) into labelled sterile 500ml Duran-Schott Bottles.
Sterility tests were conducted by aliquoting 2ml of the filtered solution from each bottle into a 15 ml centrifuge tube and left for incubation at 37 degree Celsius. The medium in the tubes were checked for pH change and turbidity after 2 to 3 days. The bottles of filtered basal medium were stored at 4 degree Celsius until completion into 4x complete medium for use.
3.3 Cell Culture
3.3.1 Cell Counting
Cell enumeration was performed using an improved Neubauer bright-line haemocytometer. The haemocytometer and cover-slip were washed with 70% (v/v) ethanol and wiped with kim-wipes. The cover-slip was placed onto the haemocytometer and ready for cell loading. A sterile 100ïl aliquot of cell culture was aseptically sampled into a 1.5ml micro-centrifuge tube using a P20 micropipette and the cell aliquot was re-suspended and a volume of 15ïl cells was obtained for cell counting. Next, 15ïl of trypan blue dye solution was mixed with the cell aliquot obtained before loading onto the haemocytometer for cell counting.
The haemocytometer was then mounted onto a light microscope at magnification of 10X and focused till its grid squares could be seen clearly. Viable cells that do not absorb the trypan blue were viewed as bright unstained cells while dead cells were stained blue. Viable and dead cells were counted in the four corners 1 mm x 1 mm grid squares. Cells touching on the bordering triple lines were only counted for either the top or the left lines. Cells touching the bottom and the right hand side lines were excluded. After counting 4 squares, the total number of cells was averaged then multiplied by the dilution factor of 2 and 104 to obtain the cell concentration (cells/ml).
Finally, the cell concentration was multiplied by the total volume of cells in the flask.
3.3.2 Cell sub-culture
According to ATCC Product Information Sheet for CRL-12357, the cell line was sub-cultured every 2 to 3 days. Firstly, the flask containing the suspension culture of T-lymphocytes was flushed thoroughly 3 times with spent medium using a sterile Pasteur pipette to remove lightly adhering cells from the surface. Cell suspension was then carefully transferred into a sterile 15ml or 50ml centrifuge tube to pellet the cells at 1300rpm for 5 to 10 minutes to obtain the cell pellet. Next, 10ml of fresh complete medium was added to disperse the cell pellet and a sterile aliquot was sampled for cell counting. Cells were seeded at 2 x 105cells/ml in complete medium into either T-25 or T-75 flasks, placed at 37 degree Celsius with 5% CO2 with cap vent and sub-culture every 2 to 3 days.
3.3.3 Cell Cryopreservation
Cells from healthy growing cultures were harvested as described earlier and frozen at 1 x 106cells/ml in freezing medium into each cryovial. Optimised rate of freezing is achieved with the use of the MrFrostyTM cell freezing container. Thereafter, cryovials were stored in liquid nitrogen until use.
3.3.4 Thawing of cells
Required number of cryovials were thawed after removal from liquid nitrogen storage and submerged partially into a water bath with shaking and as soon as the last trace of ice had vanished, the cryovial was removed from the water bath and the contents were transferred into a centrifuge tube containing 10ml of cold RPMI-1640 complete medium for centrifugation at 1300rpm for 5 minutes. The supernatant was decanted and the cell pellet was dispersed by tapping. The dispersed pellet was resuspended with 5 to 10ml of fresh medium and 100ïl of cell suspension was sampled for a cell count. The cells were seeded at the required density of 2 x 105cells/ml in a total volume of 10ml with fresh complete medium in a T-25 flask and incubate at 37 degree Celsius in a 5% CO2 incubator.
3.4 Total RNA extraction from CRL-12357
Cells were pelleted and extracted for total RNA content using QIAGEN RNeasy Plus Mini Kit (Catalogue no. 74134). Before start of experiment, micropipettes, microtube holder, bench top were disinfected with 70% (v/v) ethanol followed by RNase Zap (Ambion Catalogue no. AM9780). The centrifuge tube was flicked to loosen the cell pellet. The lysate from the cell pellet were homogenized using the QIAshredder spin column (QIAGEN Catalogue no. 79654) for 2 minutes at 14,000rpm. After homogenization, the lysate was transferred to the gDNA eliminator spin column and centrifuged for 30 seconds at 13,000rpm, the gDNA spin column was discarded. Next 600ïl of 70% (v/v) ethanol to the same volume of the lysis buffer was added to the flow-through and mixed. The RNA binding step was carried out twice. A volume of 700ïl sample was transferred to an RNeasy spin column provided in the kit and centrifuged for 15 seconds at 13,000 rpm. The flow-through was discarded and the column was capped back.
Washing step was performed three times to wash the RNeasy spin column membrane. First, 700ïl of buffer RW1 was added to the column and centrifuged for 15 seconds at 13,000 rpm. The flow-through was discarded and the collection tube was reused. Next 500ïl of buffer RPE was added to the column and centrifuged for 2 minutes at 13,000rpm. After centrifugation, the column was capped into a new 2ml collection tube and centrifuged at 13,000rpm for 15 seconds. The final washing step involved adding 500ïl buffer RPE to the column and centrifuged for 2 minutes at 13,000rpm. After centrifugation, the column was capped into a new 2ml collection tube and centrifuged at 14,000rpm for 1 minute. Lastly, elution of RNA was done by placing the RNeasy spin column in a new 1.5ml RNase free collection tube. To ensure high amount of RNA eluted, 50ïl of RNase free water added directly to the spin column membrane and centrifuged for 1 minute at 13,000rpm to elute out the total RNA. The RNA extracted was immediately put on ice and quickly measured for the concentration and the purity using an Eppendorf spectrophotometer. RNA samples were stored at -70 degree Celsius immediately to reduce degradation and prepared for subsequent Reverse Transcription synthesis of first-strand cDNA.
3.5 Measurement of Total RNA concentration and Purity in CRL-12357
Measurement of RNA concentration and purity using Nano-drop machine. The concentration reading were read in ng/ml and the absorbance reading (A260/A280) were also used as the gage for the purity.
3.6 First-Strand cDNA synthesis
Reverse Transcription reaction component on one sample
1. RNA Template
2. 2.5mM dNTPs Mix
3. random primers 3ïg/ïl
4. Oligo (dT) primer
5. 0.1M dTT
6. 5X First-strand buffer
7. SuperScript Reverse transcriptase
In each well of the PCR-strip, 3ïl of the Supermix A was pipetted. Templates were added at volumes of 10.5ml into each well and pulse spun before running in the thermal cycler at 65 degree Celsius for 5 minutes. After which, the strip was then quickly chilled on ice for approximately 1 minutes and pulse spun again to collect any sample droplets on the side of the reaction tube. In each well of the PCR strip, 6.5ml of the Supermix B pipetted into the PCR tubes was pulse spun before running in the thermal cycler at 42 degree Celsius for 50 minutes. Followed by 70 degree Celsius for 15 minutes and finally hold at infinity at 4 degree Celsius . The strip was then taken out and pulse spun to collect any sample droplets on the side of the reaction tube. The cDNA was stored at 20 degree Celsius until ready for amplification in RT-PCR.
3.7 Relative quantification Real-Time PCR
RNA concentrations measured were used for the direct estimation of cDNA concentration assuming a one to one ratio conversion of mRNA was reversed transcribed to cDNA during the reverse transcription PCR.
3.7.1 RQ RT-PCR experiment
Using ABI Fast 7500 Real-Time PCR system, the experiments were carried out with each sample in triplicates. cDNA samples acquired from the reverse transcription of mRNA were diluted to concentrations of 30 ng/ïl using 1ïl of cDNA template nuclease-free water. Forward and reverse primers for Beta-actin, IL-1ï¢, IL-6 and IFN-ï¡ï€ were diluted from the stock concentration of 100ïM to 100nM. A Supermix containing 10ïl of Fast SYBR Green Master Mix, 0.2ïl forward primer (10ïM), 0.2ïl reverse primer (10ïM) and 8.6ïl nuclease free water was made up each for Beta-actin, IL-1ï¢, IL-6 and IFN-ï¡ï€ primers respectively.
A volume of 19ïl of the Supermix was pipetted carefully into each sample well. When the run had completed, gene expression results were analyzed and saved.
Results of cell count for Exposure Assay 2 and RNA isolation of 24 hours samples.
Average number of cells = 32 / 4 = 8
Cell concentration = 8 x 104 x 2 = 160000 cells/ml
Total Cell Number = 160000 cells/ml x 25ml = 4000000 cells
Thus, to attain 4 x 106 cells for RNA isolation = 4 x 106 cells / 4000000 cells = 1ml
Average number of cells = 40 / 4 = 10
Cell concentration = 10 x 104 x 2 = 2000000 cells/ml
Total Cell Number = 2000000 cells/ml x 25ml = 5000000 cells
Thus, to attain 4 x 106 cells for RNA isolation = 4 x 106 cells / 5000000 cells = 0.8ml
Average number of cells = 112 / 4 = 28
Cell concentration = 28 x 104 x 2 = 560000 cells/ml
Total Cell Number = 560000 cells/ml x 25ml = 14000000 cells
Thus, to attain 4 x 106 cells for RNA isolation = 4 x 106 cells / 14000000 cells
Average number of cells = 95 / 4 = 23.75
Cell concentration = 23.75 x 104 x 2 = 475000 cells/ml
Total Cell Number = 475000 cells/ml x 25ml = 11875000 cells
Thus, to attain 4 x 106 cells for RNA isolation = 4 x 106 cells / 11875000 cells
Average number of cells = 26 / 4 = 6
Cell concentration = 6 x 104 x 2 = 130000 cells/ml
Total Cell Number = 130000 cells/ml x 25ml = 3250000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 325000 cells = 1.23ml
Average number of cells = 23 / 4 = 5.75
Cell concentration = 5.75 x 104 x 2 = 115000 cells/ml
Total Cell Number = 115000 cells/ml x 25ml = 2875000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 2875000 cells = 1.39ml
RNA Quantification Results for 0 hours sample using Nanodrop
Results of cell count for Exposure Assay 2 - 48 hours samples:
Average number of cells = 328 / 4 = 82
Cell concentration = 82 x 104 x 2 = 1640000 cells/ml
Total Cell Number = 1640000 cells/ml x 25ml = 41000000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 41000000 cells = 0.980ml
Average number of cells = 287 / 4 = 71.75
Cell concentration = 71.75 x 104 x 2 = 1435000 cells/ml
Total Cell Number = 1435000 cells/ml x 25ml = 35875000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 35875000 cells = 0.111ml
Average number of cells = 90 / 4 = 22.5
Cell concentration = 22.5 x 104 x 2 = 450000 cells/ml
Total Cell Number = 450000 cells/ml x 25ml = 11250000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 11250000 cells = 0.356ml
Average number of cells = 90 / 4 = 22.5
Cell concentration = 22.5 x 104 x 2 = 450000 cells/ml
Total Cell Number = 450000 cells/ml x 25ml = 11250000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 11250000 cells = 0.356ml
Average number of cells = 135 / 4 = 33.75
Cell concentration = 33.75 x 104 x 2 = 675000 cells/ml
Total Cell Number = 675000 cells/ml x 25ml = 16875000 cells
Thus, to attain 4 x 106 cells = 4 x 106 cells / 16875000 cells = 0.237ml
Results for Exposure Assay 2 - First-Strand cDNA synthesis
Results were not computed as yet as gDNA eliminator was unavailable for the 24 hours and 48 hours sample. Thus during the isolation of RNA for 24 hours and 48 hours for exposure 2, the step to eliminate the gDNA was skipped. With gDNA content along side with RNA, cDNA synthesis is ceased till stock of gDNA eliminator comes.
Results for Exposure Assay 2 - RQ RT-PCR experiment
Results need to wait till after first-strand cDNA synthesis is done and relative quantification of cDNA is done using ABI Fast 7500 Real-Time PCR system.
Chapter 6: References
Abbas, A.K. and Litchman, A.H., 2009. Basic Immunology: Function and Disorders of the Immune System. Philadelphia: Saunders.
Alam, R. and Gorska, M., 2003. 2. Lymphocyes. The Journal of Allergy and Clinical Immunology, 111(2): 476-485
J.A. Patterson, K.M. Burkholder 2007 Application of Probiotics and Prebiotics in Poultry Production [online] Available from: http://ps.fass.org/cgi/reprint/82/4/627.pdf [Accessed 9 November 2010]
Kgsmith. 2005 The Avian Immune System. [online] Available from: http://www.wingwise.com/immune.htm [Accessed 11 November 2010]
Kaiser, P., Poh, T.Y., Rothwell, L., Avery, S., Balu, S., Pathania, U.S., Hughes, S., Goodchild, M., Morrell, S., Watson, M., Bumstead, N., Kaufman, J. and Young, J.R., 2005. A Genomic Analysis of Chicken Cytokines and Chemokines. Journal of Interferon and Cytokine Research, 25(8): 467-484.
M E Rose. 1979. The immune system in birds. [online] Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1436979/?page=2[Accessed 11 November 2010]
Ritchison. G. 2001. Avian Biology. [online] Available from: http://people.eku.edu/ritchisong/birdcirculatory.html [Accessed 9 November 2010]