The overall goal of this procedure is to perform a micro RNA microarray experiment. This is accomplished by first acquiring printed microarray slides that contain a library of micro RNA probes and preparing RNA samples with suitable quality and quantity. Next, the RNA and A controlled DNA sample are labeled with fluorescent dyes.
Then the labeled nucleic acids are added to a microarray slide for hybridization. Finally, the microarray slide is washed and scanned and the slide is regenerated. Ultimately, results can be obtained that show the genome wide expression of microRNAs through analysis of the slide for hybridization intensities of the individual micro RNA probes.
This method can help answer key questions in the gene expression field, such as how normal physiological conditions or pathological conditions differential affect the expression micro needs at a global scale. The quality of microarray fabrication is one of the most critical factors for the success of a microarray experiment. After isolating RNA using a method that preserves small RNAs and resus suspending it at a concentration equal to or greater than two milligrams per milliliter, label the RNA in a 20 microliter reaction volume by first mixing about 25 micrograms of total RNA with 0.5 micrograms of five prime P-C-U-D-Y 5 4 7 3 prime in one X HEPs buffer supplemented with T four RNA Ligase 1D TT, and a TP.If less RNA is available, scale down the amount of RNA and the reaction.
If the RNA is very dilute, add a carrier such as yeast, TRNA to help with precipitation, incubate the reaction on ice inside a refrigerator for two to 24 hours. Next, add sodium acetate to 0.3 molar, and then three volumes of ethanol and precipitate the RNA overnight at negative 20 degrees Celsius. To use the slides for the first time, pre hybridize them in a filtered solution of three XSSC, 0.1%SDS, and 0.2%BSA for 30 to 60 minutes at 37 degrees Celsius.
Next, submerge the slides in water a few times. Then in isopropanol, dry the slides using a centrifuge with a slide adapter at 100 times G for five minutes at 22 degrees Celsius. Rinse lifter strips in water.
Then in ethanol before drying in air, place a slide inside a microarray hybridization chamber base and a lifter slip on top of the slide so that the lifter slip covers the probe area and its white strips. Contact the slide in the dark, spin down the precipitated labeled RNA, wash it once with 70%ethanol and air dry the pellet. The pellet should be reddish in color.
Prepare a hybridization solution using one 15th to one 100th in volume of the purified labeled reference DNA per RNA sample Dissolve the RNA pellet completely with about 60 microliters of the hybridization solution to ensure that the hybridization solution does not dry out. During incubation, add 20 microliters of water to each of the humidifying wells. In the microarray hybridization chamber base.
Add mixture of labeled RNA and reference DNA to the slide. Using a thin pipette tip, gently touch the edge of the lifter slip and through suction. Allow the solution to enter the space between the lifter slip and slide.
Place the hybridization chamber cover over the base. Then seal the chamber with metal clips. Put the entire chamber inside the plastic bag that comes with the chamber cassette.
Finally, place the chamber cassette inside a container with a wet paper towel. Then cover the container with plastic wrap and place it inside a 37 degree Celsius humid cell culture incubator for about 24 hours. To process the slides after hybridization, disassemble the chamber cassettes one by one submerge a slide, and its lifter slip in two XSSC at 22 degrees Celsius.
The lifter slip will naturally fall off the slide, wash the slides in 0.8 XSSC twice, then three times in 0.4 XSSC for a total of one to two minutes. Dry the slides using a centrifuge with a slide adapter. Next, scan the slides with a microarray scanner and use a program such as blue fuse to quantify the intensities in the image files.
Inspect individual spots to exclude abnormal spots that usually arise from pore slide printing or slide handling After hybridization. For data analysis and presentation, use programs such as gene springing and excel to reuse the microarray. Strip the slide by rinsing it in water, then immersing it in a staining dish of prewarm one millimolar NAOH and 0.1 XSSC at 62 degrees Celsius for 10 to 20 minutes.
Repeat the incubation a second time. Wash the slides several times in water for up to 60 minutes with gentle shaking at 22 degrees Celsius. Dry the slides using a centrifuge and store 22 degrees Celsius.
The slides can be used without pre hybridization. This figure shows a scanned image of a microarray demonstrating very precise and strong hybridization signals. Red spots resulted from hybridization of the reference DNA green spots from the DY 5 47 labeled RNA sample, and the yellow spots were from hybridization of both the DNA and RNA to the same probes.
The Pearson correlation coefficients between technical replicates of microarray hybridization are about 0.99 indicating excellent reproducibility. After watching this video, you should have a good understanding of how to perform a custom microray experiment, not only for quantify micronase, but also for quantify other types of nucleic acid such as mRNAs.
