Contemporary Topics 40(1)

The use of radiotelemetry in small laboratory animals: recent advances. Contemporary Topics 40 (1): 08.
Restraint of animals to enable measurement of heart rate, blood pressure and temperature causes (1) increased temperature, heart rate and blood pressure, (2) increased epinephrine and norepinephrine, (3) changes in response to drugs, and (4) decreased food intake and body weight. Among the advantages of using implantable radiotelemetry devices are (1) reduction of distress, (2) elimination of restraint stress, (3) reduction of animal use (they claim 60-90%), and (4) better data that can be collected around the clock. A brief history of the uses of telemetry is included, beginning with Maurey's experiment to measure the movement of bird wings in 1869. Other historical uses included monitoring jet pilots' ECG and respiratory rates, movement of swallowed devices in the GI tract to measure pH and pressure, the effects of space flight on dogs, and transmission of the location and temperature of woodchucks. Most of these took place in the 1950s. Since the late 1960s, miniaturization of components and improved sensors and battery power have enabled telemetry to proceed, first in larger lab animals and now increasingly in smaller species. Altogether, more than 20 types of physiologic measurements have been published, collected from more than 12 different animal species. Validation of telemetry is done to demonstrate that the data collected by radiotelemetry and conventional techniques mirror each other in both relative and absolute terms. Such studies utilize either simultaneous measurements in separate groups, or simultaneous measurements of variables by using pharmacologic or other procedures. This paper concludes that use of radiotelemetry is validated for blood pressure, ECG, and body temperature in rats and mice, based on the review of over 100 papers. Blood pressure: there may be differences between implanted sensors and acute arterial measurements, due to differences in probe design and placement. Mean arterial pressure measured by the two methods has good agreement, but systolic and diastolic measurements differ. ECG and heart rate: telemetry experiments have demonstrated that, unlike man, the mouse has no ST segment; the T wave follows immediately after the QRS complex. Ketamine anesthesia causes increased duration and variability of the RR and QT intervals in mice (at least in ion-channel defective transgenics). Treatment of mice with beta-adrenergic agonists (isoprenaline) increases the heart rate, whereas beta-adrenergic antagonists (propranolol) decreases it. Just restraining the mice to give an i.p. injection increases the rate from 400-450 to 600-650 bpm. Finally, handling lab animals that are hypertensive or normotensive increases both heart rate and blood pressure. This occurs despite training of the animals. Effects are exacerbated when rodents are handled during the daylight period. Lead placement for ECG recordings is extremely important, to minimize artifact and obtain good tracings that can be computer-analyzed. Generally, lead II configuration seems to be best, with the negative electrode placed subcutaneously near the right scapula and the positive electrode on the lower left thorax. Circadian rhythms can be monitored telemetrically using the above methods. It seems to take mice at least 5-6 days after sensor implantation to recover their normal diurnal rhythms. There was much discussion comparing the measurement of blood pressure by tail-cuff in conscious restrained animals vs. use of telemetry. Rats must be subjected to some heating to make tail-cuff measurements possible, which causes measurement errors. Some drugs have been shown to have different effects depending upon the measurement methods. Captopril (an anti-hypertensive drug) reduces blood pressure in hypertensive rats when measured using the tail-cuff method, but it has no effect when measured using radiotelemetry was used. Angiotensin-II antagonists were 10 times more potent when the effects on marmosets were measured on restrained animals vs. telemetered animals. Nitrite in the drinking water was found to decrease blood pressure in rats only at night, when measured by 24-hour telemetry methods. The authors conclude that "telemetry is the truth."
1. Restraint of laboratory animals, i.e. for physiologic measurements such as blood pressure, heart rate and ECG, has all EXCEPT which of the following side-effects?
a. decreased plasma norephinephrine and epinephrine
b. increased body temperature, heart rate and blood pressure
c. changes in responses to some pharmaceuticals d. decreased food intake and body weight
2. How long after implantation of radiotelemetry sensors does it take for mice to regain their normal diurnal rhythms?
a. 1-2 days
b. 5-6 days
c. 10-14 days
d. 21 days
3. Which of the following has NOT been shown when comparing radiotelemetric methods of blood pressure measurement to conventional methods?
a. Captopril has no effect on blood pressure in hypertensive rats when measured telemetrically
b. Angiotensin II antagonists are 10 times more potent at lowering blood pressure in marmosets with radiotelemetry
c. Nitrite in the drinking water of rats lowers blood pressure only at night
d. Systolic and diastolic pressures are comparable in rats measured with both methods
1. a. epi and norepi increase
2. b. These authors state 5-6 days
3. d. Mean arterial pressures were comparable, but systolic and diastolic differed, possibly due to differences in probe type or implant location.

The Effects of Routine Cage-Changing on Cardiovascular and Behavioral Parameters in Male Sprague-Dawley Rats. Contemporary Topics 40 (1): 17.
The authors wanted to determine if the blood pressure, heart rate, and behavior of adult male Sprague-Dawley rats are affected by the routine animal husbandry procedure of moving animals to clean cages. Those experienced with animal husbandry have noted that rats behave as though stressed after being moved to a new cage as evidenced by increased exploratory behavior, rearing, and grooming. Thus, there exists the concern that a cage change could confound experiments done subsequent to the move to a new cage.
The objectives were to change the cages and observe the following: the cardiovascular responses by radiotelemetry (transmitters surgically implanted in peritoneal cavity) and behavioral changes by direct observation; whether the responses are affected by the number of times ages are changed; whether adding a small amount of soiled bedding from the previous cage would modify these responses; and whether those animals in the room which “witness” the cage-changing activities but do not have their cages changed are effected.
Results: Systolic, diastolic, and mean arterial blood pressures; heart rate; and cage behavior (movements, rearing, grooming) increased promptly and significantly when animals were placed into clean cages.The rats which witnessed the cage-changes but were not changed themselves did not show significant increases in blood pressure, heart rate, or activity. When rats were moved to clean cages that contained new bedding plus a small quantityof the soiled bedding from their previous cage, the cardiovascular and behavioral responses were similar to those animals exposed to completely fresh bedding. The responses of rats being moved to new cages did not diminish between the first and fourth weekly cage change, and thus there appeared to be no habituation to the manipulation.
Conclusion: Ordinary animal husbandry procedures such as moving rats to a clean cage can induce transient (45 to 60 min), but significant, increases in cardiovascular and behavioral changes that could confound experiments conducted shortly thereafter.The effects of routine cage-changing on cardiovascular and behavioral parameters in male Sprague-Dawley rats
Questions:
1. How were the cardiovascular parameters measured in this study?
2. What did this transmitter measure?
Answers:
1. By implanting a radiotelemetry transmitter into the peritoneal cavity.
2. systolic and diastolic pressures, MAP, heart rate

A comparison of two opioid analgesics for relief of visceral pain induced by intestinal resection in rats. Contemporary Topics 40 (1): 21.
Authors compared the effectiveness of buprenorphine vs. oxymorphone to alleviate pain induced by intestinal resection in rats. Rats either
received 0.5 mg/kg q. 6 hours SC buprenorphine or 0.03 mg/kg q. hr. oxymorphone I.V. infusion with total parenteral nutrition (TPN). Rats
treated with oxymorphone exhibited behavior and appearance consistent with pain and distress for as long as 32 hr. post-operatively.
Additionally, despite infusion of similar TPN quantities, the volume of urine collected from rats treated with buprenorphine was decreased compared to the urinary output of rats treated with oxymorphone. Three routes of oxymorphone administration were studied: Alzet minipump, I.V. bolus injection, and continuous I.V. infusion. All three routes minimized pain-related behavioral changes associated with intestinal resection. Data from this study indicates buprenorphine is not an effective analgesic and not recommended as an analgesic for deep visceral pain associated with intestinal resection in rodents. Oxymorphone given at 0.03 mg/kg hourly is an effective analgesic for pain induced by intestinal resection in the rat.
Questions:
1. List some pain-related behaviors in animals.
2. ____________ is a partial opioid agonist; whereas
____________ is a semisynthetic pure opioid agonist.
3. T/F: Urinary retention in rodents with continuous I.V. infusion of fluids is detrimental to the animal and can impair the researcher's ability to determine fluid output and nitrogen excretion.
4. Which of the following statements concerning analgesia in rodents is TRUE?
a. Oral acetaminophen in a gelatin formulation can be used orally in rats and provides potent analgesia.
b. Buprenorphine in gelatin actually enhanced the inflammatory response and treated rats showed enhanced footpad swelling and lameness.
c. All of the above are TRUE.
5. Which of the following has NOT been used as bolus parental analgesic therapy in rodents?
a. Flunixin meglumine
b. Phenylbutazone
c. Tricyclic antidepressants
d. Morphine
e. All of the above have been used.
6. Which of the following is an appropriate form of anesthetic for neonatal rodents?
a. Inhalation anesthesia
b. Hypothermia
c. Carbon dioxide
d. A and B
Answers:
1. self-mutilation, lack of normal grooming/activity, signs of stress
2. buprenorphine; oxymorphone
3. True
4. B
5. E
6. B

Evaluation of objects and food for environmental enrichment of NZW rabbits. Contemporary Topics 40 (1): 27.
The Guide states that both structural and social environments should be considered in lab animal husbandry. Rabbits preferred pair-housing in previous studies (females; males are too aggressive) but single housing is often required in a laboratory environment. Rabbits also preferred hay as enrichment but it can pose a danger due to toxins in some grasses.
This study compared environmental enrichment in 3 groups (of six) of 6-week old NZW rabbits. The groups were:
Food enriched: Celery, Bunny stix, or Bunny block
Toy enriched: Jingle Ball, Kong, Nylabone
Control: no enrichment
The rabbits were videotaped for 1 hour daily for 15 days (via plexiglass fronted cages) and the following were quantitated:
Number of "touches" of the object
Total time spent interacting with the object
Total activity level (i.e. any time the rabbit was not lying down)
Weight gain

Results
Rabbits spent significantly more time interacting with Bunny stix than with any other food or non-food item. Bunny blox and celery interaction tapered off over time, but Bunny stix interaction actually persisted and increased throughout the study.
No significant difference was seen for interaction time or "touches" between the toys. The rabbits initially interacted with the Jingle ball and Kong but interest dropped off. They never showed much interest in the Nylabone.
Total activity was significantly greater for food-enriched rabbits. Activity did not differ between toy-enriched and control groups.
Although weight gain did not differ significantly, there was a trend toward weight gain in food-enriched rabbits. The authors suggest that a longer study might have shown a significant difference. The rabbits did not actually consume much of the food items themselves (2-4 grams of Bunny stix). Therefore, the authors suggest the gain may be a sign of well-being rather than increased caloric intake. They mention that weight gain can be problematic in rabbits held in cages long-term.
Toys such as the Jingle ball and Kong might be useful for shorter periods of time than the 15 day period of this study. Rotation of toys is suggested as a useful strategy to maintain the initial interest demonstrated in this study.
Questions
What is the genus and species of the rabbit?
What form of environmental enrichment stimulated the most activity in rabbits?
What is a useful strategy for maintaining interest in enrichment items in rabbits?
Answers
Oryctolagus cuniculus
Food
Rotation of items

Enrichment-toy trauma in a New Zealand white rabbit. Contemporary Topics 40 (1): 31.
This case report describes a potential hazard associated with the use of whiffle balls as enrichment devices with rabbits.
Case History: A 4 kg NZW rabbit was single-housed under standard housing and feed conditions. Nutritional enrichment included supplying hay and treats, including apples, pineapple sticks and breakfast cereal 2x/week.. Enrichment toys provided included plastic balls, chains, PVC tubes, woodblocks, and nylabones.
Rabbit had been housed in animal facility for three weeks without incident, until one morning animal technician found rabbit with whiffle ball stuck in her mouth and without having eaten.
Clinical Findings: Clinical exam findings showed upper and lower incisor lodged in the whiffle ball perforations. After anesthesia with ketamine/xylazine, ball was cut away from teeth. Gross clinical findings included inflamed, focally necrotic gums. Purulent exudate was present in gum tissue adjacent to incisors.
Treatment: After ball removal, gums were flushed with LRS. In addition, animal received 100ml LRS, subcutaneously to correct suspected dehydration. After full recovery from anesthesia, animal was given water by oral gavage. Analgesics were administered orally in wet feed. Soft food (wet rabbit chow and applesauce) was fed for 48 hr.. Gums appeared normal after 72 hr.
Conclusion: Several points were brought up for consideration:
1.more careful observation is necessary when introducing environmental enrichment devices Technician did not realize the ball was stuck initially.
2.Group housing settings might have further delayed recognition of problem
3.Careful safety assessment of enrichment device must be used and item should be evaluated both pre- and post-use. Animal should be monitored more carefully initially.
Safety assessments included ability to obstruct caging or watering devices, ability to withstand use without breaking, potential for broken pieces to cause damage. Further considerations included potential for physical damage, e.g. strangulation, asphyxiation or intestinal obstruction. Finally, issues such as ability to sanitize, encouragement of species-specific behaviors, and interference with research protocols must be taken into account.
Questions:
1.What is the anesthetic reversal agent used for xylazine?
2.What factors should be considered when doing safety assessments on environmental enrichment devices?
3.Give examples of situations where environmental enrichment could interfere with research protocols.
Answers:
1.Yohimbine.
2.Inherent properties of object (e.g. composition/toxicity, size, shape). Potential for harm if object is broken or damaged.
3.Examples where enrichment would interfere with research objectives:
a.use of food treats if animals on special or food-restricted diet
b.use of bells if animals being trained to respond to tonal cues
c.some enrichment devices may not be compatible with surgically-implanted or tet

Iatrogenic Horner's syndrome in an experimental pig. Contemporary Topics 40 (1): 33.
This report describes a case of Horner's syndrome in an adult domestic female pig occurring after experimental placement of an aortic stent and aortic catheterization. Nineteen days before the onset of Horner's syndrome in this animal, a woven nitinol wire stent was placed in the infrarenal abdominal aorta via the right femoral artery. Two weeks after the stent placement, aortography was performed via the right carotid artery. This procedure involved catheterizing the carotid artery, injecting the contrast media, and then ligating the artery. Five days after this procedure, the pig developed signs of Horner's syndrome: miotic pupil, ptosis of the upper eyelid, prolapse of the nictitating membrane, and enopthalmos.
The pig was kept on the study for another nine days, and then euthanized. At necropsy, a mass was located surrounding the right carotid artery, with the internal jugular vein and vagus nerve firmly attached to it. On histopathology, bacteria were identified within the mass. The vagus nerve had histopathologic changes, including mixed inflammatory cell infiltrates, and demyelination. The authors suggest that the clinical signs of Horner's syndrome in this case are consistent with the histopathologic lesions. Other possible causes for the clinical signs include general trauma to the nerve during surgery, or irritation and damage to the nerve from the contrast medium used in the angiography.
The authors stress that although this is the first reported case of iatrogenic Horner's syndrome in an experimental pig, this case should serve as a reminder for future investigators to handle these structures with care.
Questions:
1. What is the genus and species of the domestic pig?
2. What other species have had documented reports of Horner's syndrome?
3. The length of time it takes for signs of Horner's syndrome to resolve depends on the cause. True or False
Answers:
1. Sus scrofa
2. Humans, cats, dogs, horses, other large animals
3. False- Horner's syndrome due to trauma resolves fastest. Iatrogenic and idopathic causes are slower to resolve.

Restoration of fertility by transplantation of mouse ovary obtained postmortem. Contemporary Topics 40 (1): 36.
Transplantation of mouse ovarian tissue has been used to transmit genes from females who are poor breeders and would otherwise not pass on their genetic traits. In addition, cryopreservation of the ovarian tissue prior to transplant into an ovariectomized female has been shown to result in viable offspring. In this study, the authors sought to determine whether fertility would result from engraftment of ovarian tissue taken from a dead mouse and transplanted into an ovariectomized female. Tests were done on mice 0 hours to 24 hours postmortem.
Donor mice were euthanized and left in cages for 0,1,2,4,6,12,or 24 hours. The ovaries remained in situ until harvested. The mice were anesthetized with Nembutal (sodium pentobarbital). Surgical approach was from the dorsum and the abdominal cavity was incised to provide access to the ovaries. The recipients' ovaries were removed and the donor ovaries were transplanted into each empty bursal cavity. The recipient mice were housed with fertile males 14 days after surgery.
All five mice with fresh ovaries (transplanted immediately after death) delivered live pups. Two (out of five) mice receiving transplanted ovaries taken 1 hour postmortem (p.m.) delivered 5 live pups. One (out of five) mice receiving 2 hour p.m. ovaries delivered 3 live pups. All mice receiving ovaries more than 2 hours p.m. failed to conceive. PCR showed that all pups had the transgene; they all appeared to be normal, could become pregnant and deliver normally. The number of pups per litter decreased and the mean days to first litter increased by prolonging the time of extracting ovaries postmortem.
Tissue taken for transplant is usually extracted immediately and preserved in Euro-Collins (EC) or University of Wisconsin (UW) solution to prevent damage to the cells until time of use. In this study, no special medium was employed and live births still resulted from tissue taken up to 2 hours p.m. Live births can also result from in vitro fertilization of spermatozoa that are recovered from dead mice 24 hours p.m. if left at 22 C and up to 7 days p.m. if mice are kept in the refrigerator.
When transplanting ovaries from dead mice, remember that the cause of death of the donor could lead to death of the recipient (ie lymphoma in the donor can cause lymphoma in the recipient).
Questions:
1) Preservative solutions have components designed to prevent ischemic injuries such as cell swelling, free radical production, energy depletion and calcium overload. From where does the UW tissue transplant preservative solution get its name?
a. University of Washington
b. University of Wyoming
c. University of Westchester
d. University of Wisconsin
2) The following would NOT be a benefit of retrieving ovarian tissue from a dead mouse
a. rescue a valuable germline from a mouse that exhibits prepubertal lethality
b. storage of a large number of viable oocytes
c. Prevent transmission of murine leukemia virus
d. ovarian transplant into an ovariectomized female
Answers:
1) d. University of Wisconsin (my alma mater)
2) c. The virus can pass to the recipient causing lymphoma

Elevated glycemia and local inflammation after injecting N-methyl-2-pyrrolidone (NMP) into the marginal ear vein of rabbits. Contemporary Topics 40 (1): 38.
The authors of this study were evaluating N-methyl-2-2pyrrolidine (NMP) +/- Plasdone and dimethylacetate/cremophor (DMA) as vehicles that may be used to dissolve poorly soluble candidate drugs for diabetes treatment. During preliminary work, in New Zealand white rabbits, they found that NMP injection into the marginal ear vein caused hyperglycemia and local ear inflammation.
NMP, with or without Plasdone (added to eliminate local inflammation), caused hyper excitability in rabbits during bolus IV administration, and hyperglycemia for 2-4 hours post injection. Approximately 1 hour after vehicle administration, peripheral vasodilation occurred. 48 hours after NMP injection, injected ears were swollen, bluish,warm and rabbits could not hold them upright. The uninjected ear in the same rabbit appeared normal. Histologically, normal ear tissue organization was lost and the connective tissue had moderate focal necrosis and edema. Only one concentration of NMP was tested.
In the rabbits injected with the other vehicle, DMA, plasma glucose remained stable, and there were no behavior changes or lesions associated with injection.
The authors concluded that NMP should not be used intravenously in rabbits.
Questions:
Name two chemicals that can be used to induce diabetes in rabbits.
Answer:
Streptozotocin and alloxan

Hepatocystosis in baboon (Papio anubis). Contemporary Topics 40 (1): 41.
An adult wild caught East African baboon housed at the Yale Animal Resources facility was diagnosed with Hepatocystis merocysts on histopathological exam of the liver after necropsy. During the quarantine period, the animal was found to have multiple small nodules dispersed throughout the liver on radiography. Fecal, urine, and blood exams done during the quarantine period had no abnormalities. TB testing was also within normal limits. During ovariectomy surgery for the experimental protocol this animal was on, a liver biopsy was taken. Granulomatous hepatitis was found on cytology of the biopsy. Differentials included tuberculosis and chronic hepatitis due to parasites or parasitic migration. The animal was found to have hepatitis A IgG antibody at the time of ovariectomy.
The animal was euthanized 1 year after arrival and gross necropsy abnormalities were limited to hepatic changes. The liver had multiple white foci on the surface and throughout the parenchyma. There was capsular fibrosis with adhesions of the liver lobes, gall bladder, diaphragm, and liver. On histologic exam, there were multifocal degenerate and intact merocysts consistent with Hepatocystis sp. and evidence of peri-portal inflammation consistent with resolved Hepatitis A infection.
Hepatocystosis is a nonpathogenic protozoal disease endemic to Old World non-human primates. H. kochi and H. simiae are found in African primates of the genera Cercopithecus, Cercocebus, Colobus, Erythrocebus, Hylobates, and Papio. H. taiwanensis and H. semnopitheci are found in Asian primates. H. kochi is endemic to central Africa and is the only species for which the insect vector is known (Culicoides adersi). Infections with Hepatocystis is persistent but non-pathogenic as infected animals are asymptomatic and do not experience hemolysis. Since transmission requires an insect vector, it is extremely unlikely to occur in an indoor housed colony. There is no risk of transmission to humans.
Questions:
1) Name 3 of the 4 other species (besides Papio anubis or olive baboon) of baboons.
2) Which of the following statement about hepatocystosis is true:
a- Hepatocystis is a pathogenic protozoal organism endemic to Old and New World NHP, requires an insect vector, and poses no danger of transmission to humans.
b- Hepatocystis is a nonpathogenic verminous organism endemic to Old World NHP, requires an insect vector, and poses a danger for transmission to humans.
c- Hepatocystis is a non-pathogenic protozoal organism endemic to Old World NHP, requires ingestion, and poses no danger of transmission to humans.
d- Hepatocystis is a nonpathogenic protozoal organism endemic to Old World NHP, requires an insect vector, and has no danger of transmission to humans.
e- Hepatocystis is a non-pathogenic protozoal organism endemic to New World NHP, requires an insect vector, and poses a danger of transmission to humans
3) T or F - Hepatocystosis, like malaria, causes a persistent cyclic erythrocytic parasitemia resulting in fever and hemolysis.
Answers:
1) Papio papio (Guinea baboon)
Papio cynocephalus (yellow baboon)
Papio ursinus (chacma baboon)
Papio hamadryas (hamadryas or sacred baboon)
From- Nonhuman Primates in Biomedical Research- Biology and Management (ACLAM Series) 1995 p.42
2) d
3) F Hepatocystosis is persistent but non-pathogenic and infected animals are asymptomatic and do not experience hemolysis.

Unusual immunophenotype of a soft tissue sarcoma in a European polecat (Mustela putorius). Contemporary Topics 40 (1): 43.
The domesticated ferret, Mustela putorius furo, is a direct descendent of the wild European polecat, and would thus be expected to have similar types and prevalence of tumors. A 6-year-old male polecat with a 2 year history of a slow-growing abdominal mass was necropsied. The mass was fibrous, yellow-white, with a large cystic center filled with yellow serous fluid. Microscopically, the mass was characterized by an invasive anaplastic spindle cell tumor composed of pleomorphic ovoid to spindle-shaped cells arranged in haphazard bundles, sheets, and whorls. Preliminary diagnosis was poorly differentiaited sarcoma.
To further characterize the tumor, immunoperoxidase staining was done with the following results: keratin, CD34(for hematopoietic progenitor cells), desmin (intermediate filament protein in cardiac, striated, and smooth muscle), and NGFR (nerve growth factor receptor)- all neg; MSA (muscle specific antigen), SMA (smooth muscle actin), S100 (protein antigen in cytoplasm and nucleus of cells), NSE (neuron-specific enolase) all stained positive. S100 and NSE have been considered markers for neuro tissue, but have been shown to be non-specific. Likewise, desmin (for muscle) was negative, but MSA and SMA which are more specific for smooth muscle were positive, leading to a diagnosis of leiomyosarcoma. The authors stress the need for using secondary/more conserved phenotypic markers to accurately characterize poorly differentiated tumors.
Questions:
1.) What is the scientific name for the domestic ferret?
2.) What are the two most common tumors diagnosed in ferrets?
3.) Name 3 immunophenotypicmarkers for neural tissue- which is the most specific?
Answers:
1.) Mustela putorius furo
2.) adrenocortical carcinomas and lymphosarcomas
3.) S-100, NSE (neuron-specific enolase), and NGFR (nerve growth factor receptor)- Most specific is NGFR (monoclonal antibody specific for neurons and nerve sheath tumors).

Ribonuclease protection assay. Contemporary Topics 40 (1): 45.
The purpose of this assay is to quantitate simultaneously several RNA species in a single sample. Ribonuclease Protection Assay (RPA) is useful for studying and quantifying gene expression. The advantages are that it is both sensitive and specific, it allows for quantification, and can detect several RNA species simultaneoulsy. Disadvantages are that it requires large amounts of RNA from the test sample, it is not as sensitive as RT-PCR, and the procedures are tedious and time consuming. Alternative techniques are Quantitative competitive RT-PCR, and Northern blot hybidization.
Method:
1. Anti-sense probe synthesis:
Begin with a circular plasmid of RNA to be tested (target RNA) with a promotor (SP6,T7 orT3) for RNA polymerase. A complementary DNA sequence (cDNA) is inserted into the above plasmid and binds to the section of interest. The circular plasmid is cut to become linear. A RNA polymerase is added that recognizes the promotor (SP6). It makes multiple copies of cDNA-RNA sections (anti-sense RNA). A labelled nucleotide (for later hybridization reactions) is added to each above copy.
2. Probe hybridization:
Obtain a tissue sample that you wish to test. Isolate RNA Mix with the probes made in step 1. Heat to 90 degrees C. Immediately cool to 56 degrees C. This will allow anti-sense RNA probes (step 1) to hybridize with target RNA (from sample)
3. Ribonuclease Treatment:
There will be extra pieces of RNA which did not bind to the probes. Ribonucleases are added to the mixture in Step 2 to degrade single stranded RNA remaining. Only the hybridized or double stranded "protected" pieces of RNA are spared.
4 Quantitation:
Ribonucleases are inactivated with proteinase K. Hybridized RNA pieces are purified by phenol-chlorform extraction. RNA pieces are denatured and separated on a polyacrylamide gel. The quantity of target RNA is directly proportional to the amount of labelled probe.
For eg. measuring amount of radioactivity. The value is normalized to a "house-keeping" gene such as L32 or HPRT
Questions:
1. What is the purpose of RPA?
2. What are the advantages of this techique?
3. What are the disadvantages?
Answers:
1.To quantitate simultaneously several RNA species in a single sample.
2. Sensitive and specific. Quantitative. Several RNA species simultaneously.
3. Requires lg amount of RNA. Not as sensitive as RT-PCR. Tedious and time consuming.