Hi, I am Judy Y from the laboratory of Kate Rubins at the Whitehead Institute for Biomedical Research. Here we demonstrate how to label the antisense RNA samples collected from both vaccinia virus infected hela cells and virus by amino all LEO coupling of dyes. We then show how to clean up the labeled A RNA and prepare it to be hybridized for microarray analysis of gene expression.
Previous Joe videos highlight how to perform the viral infections total RNA isolation and RNA amplification and amplification steps. So let's get started. To begin a RNA labeling first add one microgram of the A RNA samples into 1.5 milliliter micro centrifuge tubes.
Vacuum dry the samples on low or no heat until they are completely dry. Cap each tube as soon as it is dry. It is important to not over dry the samples after they have dried.
Add nine microliters coupling buffer to each tube and resuspend the A RNA by gently vortexing for one minute centrifuge briefly to collect the sample on the bottom of the tube and then let the sample sit on ice. Next, add 22 microliters high quality DMSO to each tube of SCI three or S five dye one tube of dye is enough for two samples. The S SI three dies for labeling your reference samples and the SCI five dye is for labeling your test samples.
Vortex the dyes to mix thoroughly. Remember to keep the dyes in the dark until ready to use. Do not prepare the dye earlier than one hour before using and make sure no water gets in the dye DMSO mix at any point.
Now at 11 microliters of the prepared D-M-S-O-S dye mixture to each sample mixed well by vortexing. Gently incubate for 30 to 45 minutes at room temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.
After the incubation at 4.5 microliters hydroxy lamine to each sample to quench the reaction mixed well by vortexing. Gently incubate for another 15 minutes of broom temperature. Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.
To clean the label, they RNA vortex, the RNA binding beads. Briefly to obtain an even mixture before use, prepare the A RNA binding mix at room temperature. Mix the binding mix well by vortexing aliquot the A RNA elution buffer into a 1.5 milliliter tube and incubated 50 to 60 degrees Celsius for at least 10 minutes.
Add 70 microliters of the A RNA binding mix to each sample and mix well by pipe adding up and down three to four times after mixing, transfer the samples from the PCR plate to a 96 well round bottom plate. Next, add 50 microliters of a hundred percent isopropanol to each sample and mix well by pipetting up and down three to four times. Gently shake the plate on an orbital shaker for at least two minutes.
To thoroughly mix the samples. Move the plate to a magnetic stand. To capture the magnetic beads.
Leave the plate on the stand until the mixture becomes transparent and the binding beads have pelleted. Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads. Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.
The supernatant should be either a bright pink or a bright blue at this point due to the unincorporated dye molecules. Once the supernatant has been discarded, remove the plate from the magnetic stand. Now add a hundred microliters, a RNA wash solution to each well and shake the plate for one minute on the orbital shaker at moderate speed.
Beads may not fully disperse at this step, return the plate to a magnetic stand. To capture the magnetic beads. Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.
Alternatively, carefully remove the supernatant with a pipette and discard the supernatant. Remove the plate from the magnetic stand. Repeat the wash a second time with 100 microliters, A RNA wash solution.
After the second wash. Dry the beads by shaking the plate for one minute on the orbital shaker at the maximum speed. Do not over dry the samples elute the A RNA from the beads by adding 20 microliters preheated, A RNA elution buffer.
To each sample vigorously shake the plate on the orbital shaker for three minutes. Then check to make sure the magnetic beads are fully dispersed. If not, continue shaking.
Once the magnetic beads are fully dispersed, move the plate to a magnetic stand to capture the magnetic beads. The supernatant contains the cleaned up labeled A RNA samples and should be either a pale pink or a pale blue. Next, carefully transfer the alluded a RNA to a new PCR plate or PCR tubes.
At this point, you can check the RNA concentration and the amount of dye in the samples by measuring 1.5 microliters on a NanoDrop spectrophotometer. Using the microarray module immediately hybridize the labeled A RNA onto a microarray platform of your choice. Or alternatively, you can store the labeled A RNA at negative 80 degrees Celsius until you are ready for hybridization.
This is a representative spectrophotometer reading and OD plot generated using the NanoDrop spectrophotometer to measure sample concentration and dye incorporation. You can see the OD peaks at 260 nanometers for calculating the concentration of the samples as well as the two peaks around 532 and 635 nanometers representing the S3 and SCI five dyes respectively. We've just shown you how to fluorescently label amplified immuno allele incorporated RNA samples for microray hybridization.
When doing this procedure, it's important to remember to resuspend the dye in DMSO less than one hour before coupling and ensure no water gets into the dye DMSO mix as it will react with the active group on the dye. It is also important to remember not to over dry the RNA if necessary. The samples can be dried down to one to two microliters rather than completely dry, and then resuspended well in the coupling buffer during the coupling reaction.
Keep the reactions in the dark with occasional flicking and spin down if desired. So that's it. Thanks for watching and good luck with your experiments.
Be sure to watch the other videos in this series where we show how to perform a time course infection with the vaccinia virus and RNA extraction of the samples and the procedure for linear amplification of RNA samples.
