The overall goal of this procedure is to perform a microarray experiment using an automated mixer apparatus. This is accomplished by first analyzing the quality of the RNA samples with the bioanalyzer subsequent to RNA amplification and labeling. The RNA samples are hybridized to the microarrays.
Finally, the hybridized microarrays are washed and scanned. Ultimately, results are obtained that show the differential expression of RNA transcripts through analysis of dual color fluorescence microarray images. Visual demonstration of this method is critical as the microarray hybridization steps are difficult to learn because of the specialized equipment.
Quality control analysis begins with preparation of the 2, 100 bioanalyzer instrument and chip priming station as directed in the written procedure. The gel matrix is then filtered by pipetting 550 microliters into a spin filter provided with RNA 6, 000 nano kit centrifuge. The filter at 1, 500 RCF for 10 minutes at room temperature.
Then aliquot the filtered gel into 65 microliter aliquots, which can be stored at four degrees Celsius for up to one month. Vortex the dye concentrate for 10 seconds and add one microliter to a 65 microliter aliquot. A filtered gel after vortexing the mixture centrifuge at 13, 000 RCF for 10 minutes at room temperature to run the samples.
First position a chip on the priming station. In this demonstration, RNA 6, 000 nano chipps are used. Load nine microliters of the gel dye mix in the well marked with a white G against the black background.
With the pipet tip positioned at the very bottom of the well. Position the syringe plunger at one milliliter and close the priming station. Press the plunger down slowly but steadily until it is held by the clip.
After 30 seconds, release the clip and allow the plunger to rise a few seconds. After the plunger stops, pull the plunger back to the one milliliter position and open the priming station. Load nine microliters of the gel die.
Mix in each of the two wells. Mark G.Then pet five microliters of the marker into the ladder well and into each of the 12 sample wells. Next load, one microliter of the denatured ladder in the ladder well and one microliter of each denatured sample into the sample wells.
Finally, add one microliter of the marker into each unused sample. Well once loaded, vortex the chip for one minute at 2, 400 RPM. After starting the 2, 100 expert software, position the chip and close the lid.
Make sure the correct port is selected and run the assay within five minutes of loading the samples to prevent evaporation. Methods for performing RNA amplification and labeling can be found in the written protocol. Once labeled, purify the CRNA to remove any unincorporated nucleotides Using cogens r an easy mini columns, the labeled CRNA can then be quantified with a NanoDrop 2000 spectra photometer.
In microarray measurement mode, record, sample concentration, yield, and specific activity. For microarray hybridization, it is necessary to achieve a yield of at least 825 nanograms and a specific activity of at least eight picomoles per microgram at least two hours prior to the microarray hybridization. Turn on the hybridization station and set to 65 degrees Celsius.
This protocol describes the use of Agilent four by 40 4K arrays with the Roche nimble gen hybridization system and a four mixer chambers subsequent to CRNA fragmentation performed as described in the written protocol, prepare the hybridization chamber. Place a microarray slide in the assembly disassembly tool barcode side. First open an A four mixer and expose the adhesive holding the array and assembly disassembly tool to prevent any movement.
Place the A four mixer on the slide starting at the far end. Make sure it is correctly aligned to the tool and adhere the mixer along the array. Slide pull from the end of the mixer to remove the slide mixer assembly.
Using the braying tool, press all along the adhesive gasket to ensure it is tightly glued to the slide. Small air bubbles trapped between the adhesive and the slide can be seen against the dark background. Take extra time to remove these bubbles with the braying tool.
The array is now ready to be loaded. Use a positive displacement perpet to load 100 microliters of the hybridization sample. Push the tip tightly inside the porthole and dispense slowly and smoothly so the air is not trapped in the array area.
When the sample covers the whole array and liquid starts to come out the vent port quickly remove the pipette only release the plunger. Once the tip is away from the slide, gently dab the excess liquid at both ports, taking care not to draw sample out of the chamber itself. Then cover the portholes with stickers by using tweezers.
Put the slide on the hybridization station and check that the bladder holes are correctly positioned over the O rings. This will ensure proper mixing during hybridization. Close the slide cover and the station lid.
Set the station to mixing mode B and hybridize the array at 65 degrees Celsius for 17 hours. Once is finished filler glass dish labeled A with wash buffer one, the dish should be big enough to hold the assembly disassembly tool and allow some maneuvering as well. All washes should be done in glassware as plastic tends to leach compounds resulting in high background in the array.
Put a slide rack and a stir bar into a staining dish. Labeled B, fill the dish with wash buffer one covering the rack and place the dish on a stir plate at room temperature. Also, add a stir bar into an empty staining dish labeled C and place on a stir plate.
Remove the array from the hybridization station and place it in the assembly disassembly tool. Submerge the whole assembly in dish a while holding the assembly disassembly tool and the slide from opposite sides with one hand. Carefully peel off the A four mixer taking care not to scratch the array area.
Quickly place the slide on the rack and staining dish B.Exposure to the air should be minimized since the SCI five dye is sensitive to ozone. Wash the erase for one minute stirring at medium speed. Fill staining dish C with prewarm wash buffer two.
Transfer the slides and wash for one minute. After the wash very slowly, remove the slide rack from staining dish C to minimize droplets left behind on the slide. Dry the slide by spinning for two minutes.
Then place the slide into a 50 milliliter conical tube and fill with argon gas scan immediately using gene picks 4, 000 B scanner from molecular devices to avoid signal loss shown. Here are examples of four RNA samples isolated from human brain. Tumor tissue sample quality was assessed using the 2, 100 bioanalyzer on an RNA 6, 000 chip.
Solid and open arrowheads indicate the position of the 18 s and 28 S-R-R-N-A species respectively. The RNA integrity number or RIN is a quantitative measurement of RNA quality good quality Total. RNA samples should produce only two major peaks when run in a bioanalyzer for quality control corresponding to the two major ribosomal RNA species.
Some RNA degradation will show as a smear before the first ribosomal RNA peak and a significantly lower peak for 28 S-R-R-N-A severely degraded. Low quality RNA will show a broad peak or a series of peaks at low retention times. While the two ribosomal RNA peaks will be a very low intensity or not identifiable at all.
Good quality arrays should produce high signal at relatively low PMT values. Most of the transcripts are expected to be present at similar levels in the experimental and reference sample. Massive widespread changes in gene expression will probably lack any biological significance.
Therefore, most of the array should look yellow rather than green or red. Good quality signals should also be in a dynamic range such that the signal histograms fully overlap as seen in the scatterplot of the array image. After watching this video, you should have a good understanding of how to perform a microarray experiment using an automated hybridization apparatus.
