The overall goal of this procedure is to purify RNA from human retinal surgical specimens for transcriptome analysis in retinal detachment. Using journal to do this, the reagents and materials needed for collection of the surgical specimen are prepared in the laboratory and sent to the hospital. During the surgical procedure, the specimen is collected and returned to the lab.
The RNA is then purified using a standardized cesium chloride gradient procedure and the quality of the purified RNA is assessed.Ultimately. Analysis of mRNA expression shows that both inflammation and photoreceptor degeneration are involved in retinal detachment indicated by changes in expression of key genes. In these processes identified by transcriptomic analysis, The main advantage of journal over existing orna purification such as GTIN chlor from extraction, also known as ole, is the resulting of the orna are not contaminated with DNA.
The Application of this technique extend toward the therapies of retinal detachment because it provides technical means to develop novel adjuvant therapies. For each sample to be collected, use an RNAs free pipette to fill a five milliliters sterile polyethylene round bottom tube with 2.4 milliliters of a six molar RNAs free guine chloride solution. Use argon gas to fill the top part of the tubes.
Then quickly place the cap pressing tightly to ensure that it is sealed. This will prevent the oxidation of guine chloride over several years of storage in the surgical cabinet. Next, place each of the five milliliter tubes in a 50 milliliter sterile polypropylene, conical bottom tube.
After adding tissue and screwing on the cap, place the 50 milliliter tubes in a polystyrene rack. Then place the rack and prepared envelopes along with prepared shipping forms into a cardboard box in the hospital. Bring the cardboard box from the surgical cabinet to the surgical room.
During the surgery, place the retinal specimen into a tube filled with quantity and chloride solution. Close the tube tightly. Mix the tube briefly.
Then place the tube on the blood tube rocker for 10 minutes. Fill in the accompanying sample form with the patient identification surgery date and any additional remarks. Then write the identification number onto the corresponding numbered 50 milliliter tube.
Then place the five milliliter tube with the surgical specimen into the 50 milliliter tube. Replace paper tissue and cap the tube. Then place the 50 milliliter tube and sample intake form into the appropriate padded envelope and seal it.
Once the sample is received in the lab, homogenize the specimen in its five milliliter tube with a poly tron TM homogenizer at the maximum setting for one minute while moving the tube up and down. Once the sample has been homogenized, to extract the polysaccharides, add 270 microliters of two molar potassium acetate and place the tube on a shaker in the horizontal position. Shake vigorously for 10 minutes, then centrifuge for 10 minutes at 6, 500 G at 20 degrees Celsius.
Following the spin, use a pipette to carefully aspirate the soluble fraction without disturbing the pellet. Transfer the supernatant to a 14 milliliter sterile RNAs free tube. Next to the supernatant, add 5.3 milliliters of 1%and Laurel Sarcosine in 100, millimolar tri, and 3.2 grams of cesium chloride.
The sarcosine will solubilize the membrane and the cesium chloride is used to generate a gradient. Mix the tube by vortexing for one minute. Add 1.8 milliliters of cesium chloride EDTA to a sterile 11 milliliter polymer centrifuge tube.
Then with a 10 milliliter sterile pastor pipette, layer the RNA solution onto cesium chloride EDTA solution by allowing it to run down the inside surface of the tube. Place the tubes in a centrifuge. Use a second tube containing the buffers without retina as a balance if necessary, and spin for 24 hours at 225, 000 G at 20 degrees Celsius.
The next day. Following the spin, a lipid layer will have formed at the top of the tube. The DNA will be in the middle and the RNA will be pelleted at the bottom of the tube.
Use a sterile paster pipette to carefully remove and discard the upper lipid layer with a second sterile paster pipette. Gradually remove solution until the viscous DNA is aspirated. Discard the solution and DNA.
Then using a third sterile pastor pipette, remove and discard the remaining solution. Taking care not to disturb the RNA pellet. Now using a flame sterilized scalpel, cut the tube as shown here and discard the upper portion.
Place the remaining part upside down on sterile gauze. Invert the tube and use a pipette to rinse it delicately with 160 microliters of guine chloride. Let the pellet dry for 10 minutes.
Once the pellet is dry, resuspended in 150 microliters of tris, E-D-T-A-S-D-S. Next, add 150 microliters of tris, EDTA and 30 microliters of three molar sodium acetate, and then vortex to precipitate the RNA. Add 900 microliters of ice cold, 100%ethanol.
Vortex the tube and place it on melting ice for 30 minutes. Then centrifuge the tube 30 minutes at 18, 000 G at four degrees Celsius. After the spin, a tiny white pellet may or may not be visible.
Whether or not the pellet can be seen delicately aspirate the supernatant and discard it. Then to remove excess salt, add 500 microliters of room temperature, 70%ethanol and vortex, the tube centrifuge, the tube for 20 minutes at 18, 000 G at four degrees Celsius. After repeating the 70%ethanol wash and spin, use a P 200 pipette to remove any remaining ethanol.
Let the pellet air dry for 10 minutes. Resuspend the pellet in 50 microliters of dey treated water mix vigorously by vortexing for two minutes. Then place the sample in a 45 degree Celsius water bath for 15 minutes.
To fully resuspend the RNA. Finally determine the concentration of RNA by absorbance at 260 nanometers and analyze the RNA by gel electrophoresis according to the protocol. In the accompanying document, To examine the transcriptome of retinal detachment, the journal procedure was used to recover and analyze retinal RNA from 18 patients with retinal detachment and 18 age-matched controls prepared using post-mortem retinas.
RNA was subsequently purified and analyzed using gel electrophoresis and rettino base as described in this report shown here are radar graphs representing the expression of the monocyte chemotactic M CP one gene CCL two. The right part of the figure labeled RD corresponds to RNA from the retinal detachment patients, while the left part labeled N are RNA from the controls as can be seen here. Retinal detachment results in the upregulation of CCL two as indicated by the high C values in most of the RD specimens on the right compared to those of the controls on the left.
This increase indicates inflammation in the patients with retinal detachment as can be seen here. Expression of the rod photoreceptor transducing gene GNAT one was decreased contrarily to CCL two, the values are low for the RD specimens on the right, a decrease in expression of the shortwave cone opsin O PN one SW, and the homeo gene CRX was also observed suggesting photoreceptor degeneration of both rods and cones in patients with retinal detachment Through this method can provide insight into gene expression changes following retinal detachment. It can be also applied to other retinal pathology in animal models such as inherited retinal degenerations.
Individuals new to this method may struggle because it requires knowledge of nucleic acid. Cy.A visual demonstration of this method is critical as a workover, which RNA after the certification is tricky.
