RNA & DNA Extraction - QIAGEN
RNA & DNA Extraction - QIAGEN
RNA & DNA Extraction - QIAGEN
RNA is single-stranded, and it is often difficult to isolate intact RNA. RNases are the group of enzymes that degrade or denatures RNA molecules. RNA isolation therefore requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during RNA extraction and to use RNase free pipette tips and glasswares.
I extracted RNA from the theca externa and adrenal cortical cells of the obese female nulliparous Ossabaw minipigs. I used RNeasy mini and micro QIAGEN kits for my experiments. I used ethanol, several buffers like RLT, RW1, RPE, and RDD along with DNase I. I used Qubit Fluorometer and Thermo Fisher Scientific Nanodrop to quantify the RNA after extraction.
cDNA Synthesis
RNA & DNA Extraction - QIAGEN
RNA & DNA Extraction - QIAGEN
c-DNA means complementary DNA or copy DNA. Mature (fully spliced) mRNA is used as a template for preparing cDNA. In fact, cDNA can be produced from any RNA molecule. This conversion is brought about by reverse transcriptase. cDNA can be obtained both from prokaryotes and eukaryotes.
Reverse transcriptase is a RNA-dependent DNA polymerase. It acts on a single strand of mRNA. Using mRNA as a template, reverse transcriptase produces its complementary DNA based on the pairing of RNA base pairs. This enzyme executes reactions in the same way as in the DNA polymerase. It also requires a primer with a free 3′-hydroxyl group. For transcribing RNA having secondary structures, a reverse transcriptase with high temperature performance is recommended.
Real-Time PCR (RT-qPCR)
RNA & DNA Extraction - QIAGEN
Cell Culture Media & Serum Extract
Polymerase chain reaction (PCR) is one of the most useful experiments in the fields of molecular biology. It is the most fast, reliable, and affordable techniques to amplify the smaller segments of the DNA fragments.
In real-time PCR, the amount of DNA is measured after each cycle through fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules generated. This product is called an amplicon.
Data collected during the exponential phase of the reaction yield quantitative information on the starting quantity of the amplification target.
Cell Culture Media & Serum Extract
Cell Culture Media & Serum Extract
Cell Culture Media & Serum Extract
Cell culture media is one of the pivotal techniques in the Life Sciences. Cell culture media mainly includes removal of cells or clusters of cells from a sample or a living species and placing it an artificial controlled environment and study its functioning, growth, and its impact on the environment and of the surroundings.
Serum serves as an important source for proteins, vitamins, growth factors, hormones, carbohydrates, and lipids. Serum does not contain white blood cells (leukocytes), red blood cells (erythrocytes), platelets, or clotting factors.
I performed extractions for the cell culture media which were treated differently in vehicle, insulin (I), luteinizing hormone (LH) and a compound of both LH+I for the theca cells and vehicle, nanograms and micrograms of adrenocorticotropic hormone (ACTH) for the adrenal cortical cells. Whereas the serum samples were collected after ACTH and human chorionic gonadotropin hormone stimulations.
ELISA: Androstenedione
Cell Culture Media & Serum Extract
ELISA: Androstenedione
The enzyme-linked immunosorbent assay (ELISA) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones.
Androstenedione is a steroid hormone used to make testosterone and estrogen in the human body. In females, the ovaries, and the adrenal cortex releases androstenedione into the bloodstream where it is converted to produce estrogen. In males, the testis secretes large amounts of androstenedione and converts it into testosterone within the testes.
I performed ELISA for detecting and quantifying the amounts of Androstenedione in both the media for the thecal and adrenal cortical culture cells and HCG and ACTH stimulated serum samples from the obese female Ossabaw minipigs. I used androstenedione-ELISA IBL international TECAN kits for my experimentation and read at the optical density 450 nm in the ELISA plate reader.
ELISA: Progesterone
Cell Culture Media & Serum Extract
ELISA: Androstenedione
In ELISA, detection and quantification of the hormone is determined by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. The most crucial element of an ELISA is a highly specific antibody-antigen interaction.
Progesterone is the key hormone that controls the fertility and menstruation in females along with the estrogen hormone. It is secreted by the corpus luteum, a temporary endocrine gland that a female body produces after ovulation during the second half of the menstrual cycle and plays an important role in maintaining the initial stages of pregnancy.
I used Arbor Assays progesterone kits for the validation of the progesterone hormone in the theca externa cells. The interaction of the antibody-antigen was measured at the 450 nm OD in the ELISA plate reader. This analysis helped to me quantify the amount of progesterone secreted from each thecal cell culture media samples.
Thyroidectomy
Thyroidectomy
Thyroidectomy
Thyroid is a butterfly shaped bilobed gland located at the base of the neck. Histologically the thyroid gland is composed of two types of cells follicular and parafollicular cells. Thyroidectomy is recommended when a patient undergoes thyroid cancer, goiter - noncancerous enlargement of the thyroid in hyperthyroidism.
I performed a thyroidectomy in an albino mouse. The study animal model was anesthetized by using chloroform. Surgery was carried out in an aseptic condition. All the surgical instruments were autoclaved, and the operating field was disinfected with 70% alcohol. The mouse was tied vertically to the operating tray. A vertical incision was made on its neck.
Bilobed thyroid gland was visualized along with the isthmus and trachea. The thyroid gland was taken out and the skin of the animal was stitched with suture and the animal was kept under observation for its performance until it survived.
Adrenalectomy
Thyroidectomy
Thyroidectomy
The two adrenal glands are triangular , one located above each kidney in the human body. An adrenalectomy is surgical removal of either one or both adrenal glands. Benign or malignant tumors on the adrenal glands are the main causes for performing the adrenalectomy.
Albino mouse was my animal model for performing adrenalectomy. All the pre-operative measures were taken care of. A midline incision was made in the abdomen. An adrenal gland was pulled out from the anterior region of the kidney. The other tissues were placed back in the abdominal cavity.
The skin was tied with suture and all the blood shed was wiped aseptically with medical cotton swabs. Adrenalectomized mouse was regulated on saline water as it was one of the measures in the post-operative care for an adrenal ectomized animal and the animal was kept under observation until its survival.
Ovariectomy
Thyroidectomy
Ovariectomy
The ovaries are the female reproductive organ which produces female gamete Ovum. Ovariectomy is surgical removal of one or both the diseased ovaries due to ovarian cancer or ovarian cysts.
With Ovariectomy, walks in all the other associated risks like that of osteoporosis, lack of production of steroid hormones, cardiovascular disorders, and imbalance in all the other hormonal activities due to absence of a vital endocrine gland and a female gonad.
Sterilization and disinfection of all the surgical instruments and operative trays was done in 70% alcohol. Then I made an incision in the lower back of an female albino mouse with small surgical scissors. I was able to locate the ovaries in the peritoneal cavity.
Cautiously removed the ovary and sutured the muscle layer of the animal. All the post-operative care was taken into consideration and the animal was kept under lifelong surveillance.
Orchiectomy
Dissection of Male Reproductive
Ovariectomy
Testicles are the oval-shaped male gonads located in the scrotum. They produce sperms, which fertilizes a female gamete. Orchiectomy is the surgical removal of one or more testicles and is associated with numerable side effects like sterility, erectile dysfunction, low libido, gynecomastia, osteoporosis accompanied with mood swings, depression, and weight gain.
I performed bilateral castration of testes in an Albino mouse. Began with anaesthetization of the animal with chloroform, and sterilization of all the surgical instruments followed by disinfecting the operational table with 70% alcohol. I removed the hair over the scrotum of the male mice, proceeded with a midline incision in the scrotum section.
Gently removed the testis and used sterile cotton swabs for the relief from bleeding and immediately sutured the skin. Repeated the same steps for another testis. The animal was in observation for its recovery until his death.
Dissection of Male Reproductive
Dissection of Male Reproductive
Dissection of Male Reproductive
Male reproductive system includes all the organs from the pathway of a sperm from its origin in a testis to its exit in the ejaculate. Men have two testis organs located in the scrotal sac and are responsible for the production of the sperms and the testosterone hormone.
Within the testis are the coiled seminiferous tubules and within the tubules are the germ cells. These germ cells undergo meiosis to produce sperms. They are transported to epididymis where they mature.
Further, they travel to vas deferens - a canal which moves the sperm up into the abdomen and joins seminal vesicles which modifies the pH of the sperm and provides an energy source which is responsible for the contraction in the female genital tract. It then flows to the prostate gland into the urethra and then travels out through the penis.
I dissected a male Albino mouse for studying the male reproductive system, and it was a great learning experience to withstand the entire process of spermatogenesis and learn about the organs and its functioning.
Female Reproductive Dissection
Dissection of Male Reproductive
Dissection of Male Reproductive
The female reproductive system consists of a pair of ovaries, a pair of oviduct, uterus, and vagina. Ovaries are flattened ovoid shaped structure which houses the follicle. The follicle has two important functions, one of housing the female gamete - the egg/ ovum and the other is of producing the hormone, estrogen & progesterone.
The ovary sits adjacent to a large tube, fallopian tube, which opens to the peritoneal cavity and covers the ovary with a series of finger-like projections or fimbriae and transports the newly released gamete into the oviduct. If sperm is available for fertilization, it leads to the union of male and female gamete thereby, fertilization of the egg takes place. If a fertilized egg - an embryo arrives in the uterus, it will implant it.
On the other hand, if an unfertilized egg approaches the uterus, the ovum disintegrates, and the lining of the endometrium is shed which results in the monthly menstrual cycle. The cervix is the barrier between the outside world and uterus and is connected to vagina.
I dissected a female Albino mouse to study the process of oogenesis and female reproductive organs and their functions.
Vaginal Histology
Epinephrine on Liver Glycogen
Epinephrine on Blood Glucose
I studied vaginal histology of an estrous cycle in the female rodents. The estrous cycle is for 4-5 days. The vaginal epithelium undergoes several changes due to the variation in the levels of hormones in the body. Moreover, the change in the vagina is also related to the changes in the uterine lining of the female rodents.
I took a drop of 0.87% saline in the medicinal dropper and inserted it into the vaginal opening of the female albino mouse. I did it thrice to collect the vaginal secretion.
I made an even smear of the fluid on a clean slide. I allowed it to dry for a couple minutes. Then, I stained it with Giemsa stain, rinsed the excess stain with cotton balls and water and observed it under the simple and compound microscope.
Epinephrine on Blood Glucose
Epinephrine on Liver Glycogen
Epinephrine on Blood Glucose
Epinephrine is a hormone called adrenaline secreted by adrenal glands which are located above each kidney in the human body. When the concentration of the blood glucose drops down, the adrenal gland secretes the adrenaline hormone which causes the conversion of stored glycogen, in the liver, to glucose and thereby regulates the blood glucose amounts.
I used protein free sample by collecting the supernatant of the blood sample and other reagents and centrifuged them. This sample was treated with alkaline copper sulphate and lead to the formation of cupric tartrate solution.
The glucose present in the blood sample reduces to cupric oxide down to cuprous oxide thereby reducing it further to phosphomolybdic acid and forming a blue colored complex, and then was read calorimetrically.
Epinephrine on Liver Glycogen
Epinephrine on Liver Glycogen
Epinephrine on Liver Glycogen
Glycogen is the store house for the source of energy when the level of glucose in the blood is considerably low. Glycogen is accumulated in the liver in response to the insulin secreted by the pancreas and break down to glucose in response to glycogen.
Muscles and liver contain most of the glycogen which appear to be chemically similar but have marked differences in their physiological significance. Glycogen, released from the tissue when treated with strong alkali like potassium hydroxide.
Anthrone reagent and distilled water were added to the control, experimental and standard test-tubes as per the protocol and were kept in boiling water bath for next 10 minutes. The tubes were allowed to cool, and the colored complex of bluish green color was observed at 600 nm calorimetrically.
Rat Semen Analysis
Effects of Insulin Administration
Epinephrine on Liver Glycogen
I carried out rat semen analysis to evaluate the viability and mobility of the sperms by microscopic examination using Eosin-Nigrosin staining technique. The staining solution was prepared in our laboratory with sodium chloride and sodium hypophosphite.
I took 1 ml of semen sample, added it to twice the quantity of staining solution and kept it at room temperature for about an hour. I observed, the non-viable cells were penetrated by the eosin stain and appeared red in color whereas, the viable cells were not stained.
Nigrosin facilitated a dark background for the detection of viable cells. With the help of WBC pipette, I loaded a drop of semen sample on a Neubauer chamber and allowed it to settle for 5 minutes. Finally, I was able to observe the sperms and count the number of viable, non-viable, mobile, and immobile sperm cells in each square.
Induction of Polycystic Ovary
Effects of Insulin Administration
Effects of Insulin Administration
Polycystic ovarian syndrome is a most common endocrine disorder which affects the reproductive system of women in their child-bearing ages. Polycystic ovarian disorder is characterized by hyperandrogenemia.
Hyperandrogenemia and abnormal levels of other sex hormones prevents normal menstrual cycle due to the lack of growth of small follicles to become a mature and dominant follicle and release an ovum in the ovary.
I treated a female albino mouse with an effective drug to induce polycystic ovarian syndrome. After a couple weeks the ovary was observed. I used microtome to prepare permanent slides for observing the cells of polycystic ovarian syndrome. I studied these cells under compound microscope for histological and cytological studies in this regard.
Effects of Insulin Administration
Effects of Insulin Administration
Effects of Insulin Administration
Insulin is a hormone secreted by pancreas to balance the blood glucose levels. After the intake of one's meal, the food that contains carbohydrates get converted to glucose. Most of the glucose travels into the blood via bloodstream and is one of the leading factors for the rise in the blood glucose levels.
Thus, increase in the blood glucose levels triggers the beta cells of the pancreas to secrete insulin into the blood to regulate the glucose level in the blood. If the glucose amount in the blood is unregulated it will cause diabetes mellitus.
I studied the effect of insulin administration on blood glucose level in albino mouse and was flabbergasted by the functioning and significance of Insulin in the human body.
Determination of Cholesterol
Determination of Cholesterol
Determination of Cholesterol
Cholesterol is one of the important building blocks of the hormones such as, estrogen, testosterone, and adrenal hormones. Cholesterol produces bile acids, which helps the body in digestion of fats & absorption of important nutrients. I learnt cholesterol esterase hydrolyses cholesterol esters to free cholesterol & fatty acids.
Free cholesterol is oxidized to cholestene-3-one and hydrogen peroxide by cholesterol oxidase. Peroxidase catalyzes the reaction of hydrogen peroxide with 4-aminoantipyrine, and phenol is catalyzed by peroxidase.
This generated a red colored red quinoneimine derivative which was measured at 500nm. I also studied the causes and several diseases associated with Hyper and Hypo cholesterolemia.
Estimation of Proteins
Determination of Cholesterol
Determination of Cholesterol
Proteins are the most abundant organic molecules of the living system. They are the fundamental basis of structure and function of life. Proteins are the polymers of amino acids.
I performed the estimation of proteins from the given sample by Peterson-Lowry method which is used to determine the concentration of protein in a solution by the exhibition of color change. The protein is treated with alkaline copper sulphate, where the peptide bonds of proteins produce divalent copper ion which is further reduced to monovalent ions.
These monovalent ions then react with the Folins reagent to give a blue colored complex due to oxidation of aromatic amino acids.
Estimation of Amino Acids
Determination of Cholesterol
Determination of Creatinine
Amino acids are group of organic compounds containing two functional groups amino and carboxyl. I used the Ninhydrin reagent - a powerful oxidizing agent to estimate the number of amino acids.
In the presence of Ninhydrin reagent, amino acid undergoes oxidative deamination liberating ammonia, carbon dioxide, a corresponding aldehyde. The ammonia reacts with Ninhydrin to give a bluish-purple colored complex.
The Beer-Lambert law states that the amount of light absorbed is proportional to the number of molecules of absorbing substance in the light path.
The absorption is proportional both to the concentration of the sample solution and to the length of the light path through the solution.
Determination of Creatinine
Determination of Creatinine
Determination of Creatinine
Elevated levels of creatinine are found in acute kidney failure, chronic kidney insufficiency and hypo perfusion of the kidneys, congestive heart failure, diabetes acromegaly. Decreased levels are found in muscular dystrophy. Creatinine emanates in plasma and serum and to a smaller extent in urine.
I carried out the determination of creatinine in serum by Jaffe's reaction, where creatinine reacts with picric acid in an alkaline medium. An alkaline environment is provided by sodium hydroxide. The yellowish orange colored complex is developed within 15 minutes at the room temperature and is measured at 520 nm.
Chromatography
Determination of Creatinine
Electrophoresis
Some materials appear homogeneous, but, are a mixture of few or more components. Chromatography is a technique which enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Chromatography is a versatile technique which uses stationary phase or mobile phase.
Chromatography has several applications in the fields of DNA fingerprinting, bioinformatics, forensics testing, immunization, food industry, pharmaceutical companies, environmental agencies, biomedical applications.
I performed thin-layer chromatography for identification of lipids, adsorption column chromatography for separation of pigments from flowers, ion-exchange chromatography for separation of amino acids and 2D paper chromatography for identification of amino acids and for the separation of the gonadal hormones.
Electrophoresis
Determination of Creatinine
Electrophoresis
Electrophoresis is a process where the migration and separation of charged particles (ions) takes place under the influence of an electric field. An electrophoretic system consists of two electrodes of opposite charge (anode, cathode), connected by a conducting medium called an electrolyte.
I performed sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins based on their molecular weight and studied that smaller proteins travel faster due to lack of resistance from the gel matrix used in electrophoresis.
In this type of electrophoresis, the structure and charge of proteins is highly eliminated, and proteins are separated solely based on polypeptide chain length.
Erythrocyte (RBC): Total Count
Leukocytes (WBC): Total & Differential Count
Leukocytes (WBC): Total & Differential Count
Firstly, clean the RBC pipette with distilled water, then with absolute alcohol, further with ether and keep it dry. Take a small amount of diluting fluid in the watch glass and keep it aside.
Draw blood to the 0.5 mark in the RBC pipette without letting any air bubbles into the pipette. Draw the diluting fluid to the mark 101. Then thoroughly mix the blood and the diluting fluid in the pipette by gently rolling the pipette horizontally.
Take the hemocytometer and place a clean and dry cover slip on the counting chamber. Allow a small drop of diluted blood enter the counting chamber with the help of capillary action.
Allow it to rest for approximately 3-5 minutes for the cells to settle. Later, focus the RBC counting chamber under high power microscope. Preferably, 4 corner squares and 1 central square; each having 16 smallest squares; should be counted to obtain a satisfactory value.
While counting each small square, cells touching the top and left margin should be avoided and the cells touching the bottom and right margin of each square should be counted.
Leukocytes (WBC): Total & Differential Count
Leukocytes (WBC): Total & Differential Count
Leukocytes (WBC): Total & Differential Count
A white blood cell count measures the total number of WBC in the blood, and a WBC differential count determines the percentage of each type of WBC's present in the blood. There are five types of white blood cells namely, neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
Blood should be carefully drawn, without any air bubble. WBC reagent should be drawn to 11 mark to dilute the blood. The blood and the diluting WBC reagent should be mixed by shaking the pipette vigorously for 2-3 minutes.
The tip of the pipette is touched to the Neubauer counting chamber and a drop of the fluid will sweep into the counting chamber. Wait for 3-5 minutes, allowing white blood cells to settle down.
Total number of cells in 4 squares at the corner of counting chamber is determined under the low objective of the microscope.
To identify different types of white blood cells, a thin blood film is made on a glass slide. Different cells based on their physical and structural properties are manually counted thus, gives the percentage of different white blood cells.
Estimation of Hemoglobin
Leukocytes (WBC): Total & Differential Count
Estimation of Blood Glucose
Estimation of hemoglobin is routinely and frequently performed screening hematological test of laboratory services. Hemoglobin concentration provides information about the status of anemia in the population.
When the blood is added to N/10 Hydrochloric acid the hemoglobin present in blood is converted to acid hematin which is a dark brown colored compound. The acid hematin complex is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is then estimated by reading the value directly from the scale.
With the help of a pricking needle, prick the tip of middle or ring finger. Suck the blood from in Hb pipette up to the mark of 20 µl. Add this blood sample to the acid solution which is placed into Sahli’s graduated tube. With the help of a glass stirrer, mix it and allow the tube to stand at room temperature for 10 minutes.
Gradually, add distilled water, drop by drop, into the mixture placed in Sahli’s graduated tube, till the color of the solution matches that of the brown glass standard. Take the reading of the lower meniscus from the Sahli’s graduated tube.
Estimation of Blood Glucose
Estimation of Blood Glucose
Estimation of Blood Glucose
Blood glucose is the sugar, that the bloodstream carries to all the cells in the body to supply energy. Blood glucose measurement represents the amount of sugar being transported in the blood.
The aldehyde group of glucose condenses with ortho-toluidine in glacial acetic acid. Further, on heating it gives a green color which is measured calorimetrically at 620nm.
Take 3 test tubes and marked them as blank(B), standard(S), test(T). To each test tube add 10ml of ortho-toluidine reagent. Add 0.1ml of working standard solution to the test tube marked as (S). To the tube marked as (T), add 0.1ml of serum test solution and add 0.1ml distilled water to the test tube labelled as (B).
Put all the test tubes in boiling water bath. Cover the contents of the test tubes for heating with the help of cotton balls. Boil the test tubes exactly for 5-8 minutes. After the timer goes off remove the test tubes and allow it to cool in the room temperature. The intensity of color is proportional to the glucose concentration.
Chick Embryology
Estimation of Blood Glucose
Human Electrocardiography
Chick embryology is the study of development of the chicken inside an egg. Studying chick embryology helps one to identify possible causes of embryo mortality during incubation.
I observed a wholemount preparation of the chick embryo at various hours:
18 hours: The primitive streak was lying in the middle area pellucida as a clear line, and ends in a small pit, called Hensen's node. The notochord was formed and can be distinguished now. This structure induced the formation of the neural plate and later formed the neural groove.
36 hours: The extra embryonic area had grown. Hensen’s node had given rise to 13-14 pairs of somites. The brain was differentiated into fore brain, mid brain, and hind brain. Cardiac vesicle had given rise to the heart.
72 hours: Heart had been differentiated into ventricle and atrium. The eye had developed an optic cup, lens, and an optic vesicle. A pair of fore limb buds and hind limb buds had been developed, which will develop into fore limbs & hind limbs, respectively. Moreover, the olfactory pit, visceral arches, amnion, allantois and amniotic cavity have also developed by this period.
Human Electrocardiography
Estimation of Blood Glucose
Human Electrocardiography
Electrocardiography is the process which records the electrical activity of the heart. The overall goal of performing an electrocardiography is to obtain the information about the structure and function of the heart.
An electrocardiograph is a machine that is used to perform electrocardiography and produces the electrocardiogram.
A normal heart rhythm contains a P wave, a QRS, and a T wave. The normal amplitude, deflection, and duration of each component is essential in knowing the accurate rhythm and helps in the interpretation of human electrocardiography.
P Wave has an amplitude of 2-3 mm high with the duration of 0.06 - 0.12 seconds.
PR Interval constitutes 0.012 - 0.20 seconds. QRS Complex has an amplitude of 5-30 mm high and lasts for 0.06 - 0.10 seconds.
ST Segment lasts for 0.08 - 0.12 seconds.
T Wave has an amplitude of 0.5 mm in limb leads and has the duration: 0.1 - 0.25 seconds.
QT Interval should be in between 0.36 - 0.44 seconds.
Stages of Mitosis
Stages of Mitosis
Stages of Mitosis
Mitosis is the process of nuclear cell division, which results in two daughter cells having identical number and same kind of chromosomes.
Mitosis specifically refers to separation of the duplicated genetic material that houses in the nucleus. There are four stages of Mitosis: Prophase, Metaphase, Anaphase and Telophase followed by Cytokinesis.
To study the stages of mitosis, I performed an experiment with the help of freshly sprouted onion root tip. Added 2-3 drops of acetocarmine stain to the tip of the sprouted onion root, as it stains the chromosomes and mitotic division can be visualized with the help of a compound microscope.
Stages of Meiosis
Stages of Mitosis
Stages of Mitosis
Meiosis is a division process which divides a diploid cell (one with two sets of chromosomes) to haploid cell (ones with single set of chromosomes).
Meiosis is a two-step division process. homologue pairs separate during a first round of cell division, called meiosis I. Sister chromatids separate during a second round, called meiosis II.
I performed temporary squash preparation of testis of grasshopper (Acridomorpha), to study the phases of meiosis. Initially, I observed the slide in low power objective and then gradually switched to high power objective lens.
Human Karyotype
Stages of Mitosis
Human Karyotype
I studied the structures of chromosomes in human karyotype. I learnt numerable metabolic disorders associated due to absence of one chromosome or due to its missing or additional part.
I also studied varied banding techniques, which helped me in classification, recognition of the primary chromosomal structural changes. The DNA molecule stores all the biological information and is packed into thread-like strand called chromosomes.
Each chromosome has a constriction point called the centromere, whose location on each chromosome gives the chromosome its characteristic shape and can be used to help describe the location of specific gene.
DNA Estimation
Blood Groups & Rhesus Factor
Human Karyotype
DNA is deoxyribonucleic acid. It is the house of all the genetic information. DNA is a hereditary material comprised of 4 chemical bases namely, adenine, guanine, cytosine, and thymine.
I studied the estimation of DNA by diphenylamine method, where diphenylamine reacts with deoxyribose sugar under an intense acidic condition which leads to depurination of the DNA & causing dehydration of deoxyribose sugar thereby leading to the formation of a blue colored complex. The color intensity was measured using a red filter at 595nm.
RNA Estimation
Blood Groups & Rhesus Factor
Blood Groups & Rhesus Factor
RNA stands for ribonucleic acid. The main function of RNA is to carry the information of the amino acid sequence from the genes to the assembled proteins on the ribosomes in the cytoplasm. There are 3 types of RNA namely, mRNA, tRNA and rRNA.
I studied the estimation of RNA with the help of orcinol method, a reagent which is used in chemical tests for pentoses. The pentoses leads to formation of furfural when heated with hydrochloric acid and orcinol reacts with the furfural in the presence of ferric chloride, which acts as a catalyst to give a green colored complex.
Blood Groups & Rhesus Factor
Blood Groups & Rhesus Factor
Blood Groups & Rhesus Factor
There are four major types of blood groups in the human society and is determined by the presence or absence of two key antigens, A or B on the surface of the red blood cells.
Additionally, there is a protein called the Rh factor which can either be present, making it positive or absent leading to negative. Thereby, creating 8 most common types of blood groups namely: A+, A-, B+, B-, O+, O-, AB+, AB-.
We determined the blood groups along with Rh factors for nearly 06-07 blood samples. Later, we were asked to prick ourselves and derive the blood group and Rh factor of our own blood.