This procedure starts with CDNA that has been synthesized from a template of high quality RNA. The CDNA is first labeled with the fluorescent dye SI three. Upon completion of the labeling reaction, the labeled CDNA is purified, isolated and the quality of the labeling reaction assessed.
The labeled CDNA is then hybridized onto a microarray. Following hybridization, the arrays are analyzed to determine relative levels of gene expression in the samples. Hello, my name is Sam Peterson from the laboratory of Dr.Jennifer Freeman in the School of Health Sciences at Purdue University.
Previously, we have shown you how to extract RNA from zebrafish embryos and turn that RNA into CDNA. Today we'll show you procedure for labeling and hybridizing that CDNA onto a zebrafish, oligo nucleotide microarray platform to study global gene expression profiles. Typically, we use this procedure to study differential gene expression in response to an exposure to a toxin.
So let's get started. The microarray experiment requires a minimal light environment to prevent photo degradation of the fluorescent dye SI three used in labeling to start labeling the CD NA with sci three haw the following components of the bio prime array CGH genomic labeling system 2.5 x random primer buffer, 10 X-D-C-T-P mix and nuclease free water. In addition, thaw the one millimolar stock of the SI three labeled DCTP after thawing, briefly centrifuge or reagents in a nought 0.6 milliliter thick walled tube combined.
500 nanograms of CDNA 40 microliters, 2.5 x random primer buffer and nuclease free water to achieve a total volume of 86 microliters. Mix well and briefly centrifuge, then incubate the reaction at a hundred degrees Celsius in a thermocycler for five minutes. At the end of the incubation, immediately transfer the reaction into an ice bath slurry for 10 minutes.
Next, take the exo DNA polymerase outta the freezer and place on ice. Then add to the reaction tube, 10 microliters of the 10 X-D-C-T-P Mix two microliters of the one millimolar SI 3D CTP and two microliters of the XO cleanout DNA polymerase mixed well and briefly centrifuge. Finally incubate the reaction at 37 degrees Celsius for 16 hours and a thermocycler when the labeling is finished.
Proceed to clean up and precipitate the labeled CDNA. To begin cleaning the labeled CDNA. Place a MicroCon column into 1.5 milliliter tube.
Add 300 microliters nought 0.1 XSSC to the column and then add the labeling reaction. Centrifuge the column at 16, 100 G for 10 minutes at four degrees SIUs. Notice that the flow through may be light pink.
Discard the flow through and add another 300 microliters of nought 0.1 XSSE to the column. Then repeat the centrifugation at 16, 100 G for 10 minutes at four degrees Celsius. Again, discard the flow through and add an additional 300 microliters T 0.1 XSSC to the column now centrifuge at 16, 100 G for 12 minutes at four degrees Celsius after this last wash.
The flow through should be clear and the pink labeled CDNA is visible on the column. Again, discard the flow through from this final wash to elude the labeled CD NA.Add 100 microliters nuclease free water to the column. Take the column outta the micro centrifuge tube and inver inverted into a new tube.
Then centrifuge the inverted column at 2, 300 G for two minutes at four degrees Celsius. The labeled CDNA is dissolved in the water at the bottom of the tube. Check the alluded CDNA using a NanoDrop ND 1000 spectrophotometer according to the manufacturer's instructions.
Calculate the DNA concentration and evaluate dye incorporation as described in the accompanying written protocol. If the CDNA is sufficiently labeled, put six micrograms into a new tube to precipitate the labeled CD NA mix in naut 0.1 x volume of three molar sodium acetate at pH 5.2 and one x volume. Isopropanol incubating the dark for 30 minutes at room temperature.
Keeping in mind all steps to this point have been carried out in a minimal light environment. Then centrifuge the sample at 16, 100 G for 20 minutes at room temperature. After centrifugation, carefully discard the SUP natant without disturbing the DNA pellet, which is pink.
Finally washed by adding 500 microliters, 70%ethanol and mixed by gentle inversion centrifuge at 16, 100 G for five minutes. At room temperature, remove the supinate, invert the tube, and allow the pellet to dry for five minutes. The labeled CDNA is ready for hybridization.
Before starting the hybridization, turn on the nimble gen hybridization system and allow it to equilibriate to 42 degrees Celsius. Start by dissolving the labeled CD NA pellet in five microliters of nuclease free water to the dissolved pellet. Add hybridization components from the Roche Nimble Gen Hybridization kit Hybridization buffer hybrid A and alignment oligo to a total volume of 18 microliters as outlined in the accompanying written protocol.
Mix well and briefly spin the tube to collect the contents at the bottom. Next denature, the labeled CDNA by placing the tube at 95 degrees Celsius for five minutes. Immediately transfer the tube to the nimble gen hybridization system until the arrays are ready.
Note that the microarrays require careful handling and storing. Roche nimble gen arrays are stored in the dark with desiccant at room temperature. Load the array into the precision mixer alignment tool.
The PMA T load in X one mixer onto the PMA T and remove the adhesive strip with forceps. With the array and mixer loaded, close the PMA T securely. Next, disengage the array and mixer from the PMA T by applying pressure to the mixer through the hole in the PMA t.
Finally, use a brayer to apply pressure around the outside of the mixer in order to make sure that the array is sealed and to remove any bubbles. Next place the array mixer complex onto the hybridization system to warm, to load the CDNA sample onto the array. Use the displacement perpet slowly and with steady pressure.
Add 15 microliters of the labeled sample into the fill port in the center of the mixer. Wipe off any excess sample from the port holes and seal them with adhesive strips. Then close the latch.
Now start mixing the hybridization solution using program B on the nimble gen hybridization system. Allow the microarray to hybridize for 16 to 20 hours After the hybridization, the microarray will be washed and scanned when the hybridization is nearing completion. Prepare for scanning by turning on the gene picks 4, 000 B array scanner and let it warm up for about 15 minutes.
Next, prepare wash buffers one A, one B two, and three as outlined in the accompanying written protocol. Pre-war wash buffer one A to 42 degrees Celsius, but leave the other buffers at room temperature. When wash buffer one A has warmed up, pour it into a dish.
Submerge a slide rack in wash buffer one B.Now remove the array mixer complex from the nimble gen hybridization system and slide into the jig. Start the washing by submerging the assembly into the prewarm wash buffer one A and carefully remove the mixer by peeling away from the slide. Then remove the array from the jig and agitate for 15 seconds in wash buffer one A.Quickly transfer the array into a slide rack submerged in wash buffer one B and agitate the slide rack vigorously for two minutes.
Then transfer the slide rack into wash buffer two and agitate for one minute. Lastly, transfer the slide rack into wash buffer three and agitate for 15 seconds. Finish the washers by transferring the array into the slide drying centrifuge and spin for two minutes following centrifugation.
Scan the array immediately since the D is sensitive to light and ozone, start the scanning by loading the array barcode down into the gene picks 4, 000 B array scanner. In the hardware setting dialogue box set, the 532 nanometer laser has a PMT value set at 500, set the power to 100%pixel size to five microns, lines to average to one and focus position to naught microns. When setting up is complete, preview the array.
Choose an area to scan and then scan it. Once scanning is complete, check the image histogram. Ideally, one E minus five normalized counts should be at 65, 000 saturation limit, which means that approximately one to 2%of the features are saturated.
If the normalized counts are less than one E minus five, increase the PMT gain and re-scan the slide. If the normalized counts are greater than one E minus five, decrease the PMT gain and re-scan the slide, save the image, and use a data analysis program for data normalization and calculation of intensity values. The labeling reaction routinely yields at least 10 micrograms of labeled CDNA.
Dye incorporation can be calculated using a simple base dye ratio where the nucleotide to SI three ratio equals the A two 60 to a five 50 ratio multiplied by 23 point 15. A good reaction will result in a value between 50 and 80. An acceptable hybridization will have one to 2%of the features saturated yielding.
One E minus five normalized counts at the 65, 000 saturation limit. With the PMT value set, near 500 hybridization quality can also be qualitatively assessed by viewing the scanned image of the microarray and assessing the intensity of the aligned oligos to other features on the array. The scanned image should also be assessed for any particular or dust spots within the array area.
In subsequent analysis, a scatter plot can be used to image your analyze data to determine relative levels of gene expression in the samples and to identify genes that are differentially expressed. Genes that are differentially expressed will deviate significantly from the best fit line In a complimentary procedure. We've shown you how to extract RNA from zebrafish embryos into synthesize c CD NA.Here we just showed you how to label and hybridize CD NA onto an oligonucleotide microray platform to study global gene expression profiles.
When doing this procedure, always remember to use high quality CD NA and avoid exposing the site three dye to direct lighting. So that's it. Thank you for watching and good luck with your experiments.
