Hi, I am Judy Yen from the laboratory of Kate Rubins at the Whitehead Institute for Biomedical Research. After infecting hela cells with vaccinia virus and isolating total RNA, shown in a previous Joe video protocol post and viral RNA can be isolated and amplified using reverse and in vitro transcription, the amplified RNA will be used later in microarray analysis of viral and host gene expression. So let's get started.
To begin this c cd NA synthesis from the RNA add the recommended volume of a hundred percent ethanol to the wash buffers before using the ambient amino allele message AMP two kit in a PCR reaction tube, add between 100 nanograms to five micrograms of total RNA and one microliter of T seven oligo primer. Bring the volume up to 12 microliters with nuclease free water. Incubate the samples at 70 degrees Celsius for 10 minutes in a thermocycler.
Next, remove the RNA samples from 70 degrees Celsius in centrifuge. Briefly, after centrifugation, place the samples on ice. Prepare the first strand synthesis master mix with a 10%volume overage to cover pipe editing error.
Leave it at room temperature gently pipette master mix or flick to mix, and then centrifuge briefly after centrifugation, transfer eight microliters of the master mix to each RNA sample mixed by pipe adding up and down three to four times incubated 42 degrees Celsius for two hours in thermocycler. After incubation centrifuge the samples briefly in place on ice. Immediately proceed to the next step of DS dsd A synthesis.
Prepare the second strand synthesis master mix on ice with a 10%volume overrate to compensate for pipetting error gently pipette master mix or flick aix and then centrifuge briefly after centrifugation. Transfer 80 microliters to each sample and gently mixed by pipetting up and down three to four times. Incubate at 16 degrees Celsius for two hours in the thermocycler.
After the two hour incubation, proceed with the CDNA cleanup step or freeze immediately a negative 20 degrees Celsius. To begin double stranded CD NA cleanup. First, remove the cDNA pure the refrigerator and allow it to equilibrate to room temperature for 30 minutes before use, shake the bottle to fully resuspend the magnetic CD NA binding beads prior to use aliquot nuclease free water into 1.5 milliliter tube and incubate at 50 to 60 degrees Celsius for at least 10 minutes.
During the previous two hour incubation, add 180 microliters of CD NA pure to each sample and thoroughly mixed by pipe adding up and down. Transfer the samples to a 96 well round bottom plate. After the samples have been transferred to a plate, continue to mix the samples by gently shaking the plate on an orbital shaker for at least two minutes.
Move the plate to a magnetic stand to capture the magnetic beads. Leave the plate on the stand for approximately six minutes or 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. After the supernatant has been discarded, remove the plate from the magnetic. Stand next at 150 microliters, CDNA wash buffer to each well and shake the plate for one minute on the orbital shaker at moderate speed.
Beads will not disperse at this step due to the low surface tension of the wash buffer. After shaking, 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.
Or 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 150 microliters, CDNA wash buffer.
After the second wash, dry the beads by shaking the plate for two minutes on the orbital shaker at the maximum speed. It is important to not let the beads over dry. Next elute the CDNA from the beads by adding 18 microliters of the preheated nuclease free water to each sample vigorously.
Shake the plate for three minutes on the orbital shaker. 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, carefully transfer the eluded cd NA to a new PCR plate or PCR tubes. You should be recovering approximately 16 microliters of the elucian per.
Well proceed directly to the next step or freeze the cd NA at negative 20 degrees Celsius. Prepare the IVT master mix at room temperature. Gently pipette the master mix or flick to mix and centrifuge briefly.
After centrifugation, add 24 microliters to each sample and gently mixed by pipetting up and down three to four times incubated, 37 degrees Celsius for 14 hours in a thermocycler. Then hold at four degrees Celsius until ready for the next step, vortex the RNA binding beads briefly to obtain an even mixture before use. Prepare the A RNA binding mix at room temperature.
This can be done ahead of time. The prepared binding mix can be stored at room temperature for up to one week. Mix well by vortexing aliquot the A RNA elution buffer into a 1.5 milliliter tube in incubate at 50 to 60 degrees Celsius for at least 10 minutes.
Now, add 70 microliters of the A RNA binding mix to each sample and mix well by pipetting up and down three to four times. 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. After the beads have pelleted, carefully aspirate the supernatant with the 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. Next, add 100 microliters a RNA wash solution to each well and shake the plate for one minute on the orbital shaker At moderate speed, the beads do not have to fully disperse. At this step, move 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.
Once the supernatant has been removed, move 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.
Remember to not over dry the samples elute the A RNA from the beads by adding 40 microliters of the 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 have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads. The super natan contains the cleaned up amino allele, incorporated a RNA.
Carefully transfer the eluded a NA to a new PCR plate or PCR tubes. At this point, you can check the RNA concentration of the samples by measuring 1.5 microliters on a NanoDrop spectrophotometer. Immediately continue on to the next step or store the A NA at negative 80 degrees Celsius.
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 that doing a second round of amplification is not advised. As biases and array data have been observed, it's also important to remember that mixing carefully at each enzymatic step suggests the first and second strand.
CDNA synthesis, or the IVT step is critical to obtaining good amplification yields, as is incubation of each enzymatic step at the appropriate temperature. A PCR cycler with an adjustable heated lid is preferred as even deviations as small as two to three degrees during the in vitro transcription. In an air hybridization oven or water bath hybridization can significantly affect the yield of the amplified product.
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 preparation and cleanup of fluorescently labeled RNA.
