Hi, my name is Ena and We routinely do gene expression profiling in the lab. And we use this technique to monitor differential regulated genes between meth and wild type. And today I have a wild type and meth RNA samples and we all do C first.
We use a indirect labeling methods and the advantage of this technique is to prevent eye bias that can occur in direct labeling. And to do that we have RNA samples and we opt obtain three microgram of RNA in 13 microliter of to total volume. And we add 2.5 micro of random he in our parks.
And we'll incubate sample at seven five degree to have to the nature of the RNA for eight minutes. And now we'll add the, we'll add reverse transcription cocktail that contains AM L-D-L-D-U-T-P continu mix, reverse transcriptase buffer and also ET TT Big it down And incubate the sample at 42 degree for four hours. So after four hours we just take the samples out.
And now in the tube we have CDNA and RNA mix. And what we want to do is do hydrox RNA by adding sodium hydroxide and EDTA and final concentration for sodium hydroxide should be a hundred molar. And final concentration for e BT should be five molar.
And now we will incubate samples at 65 degree water bath for 10 minutes. So the incubation is done and we'll utilize samples by adding pH seven heaps one water to the final concentration of 500 molar. At this point you can store the samples at minus 20 or you can continue with the cleanup.
For cleanup of the CDNA, we use KaiGen PCR purification kit. However, since Bush buffer and ion buffer contains freeing, we don't, we prepare our own fate bush buffer and fate ion buffer and you'll add 300 liter of PPI and we'll pipe that pond down slowly to you will transfer the mixture into columns And you'll spin for one minute at the highest speed And you'll watch the, the buffer that we prepared. Phosphate wash buffer, you'll add 750 microliter and so, and you'll add another For one again.
So we'll spin again just to make sure that we got all bed all. And we will transfer these tubes into empty offender tubes. And I'll add 30 markers of pho phosphate E buffer, which is four four molar of potassium phosphate at pH 8.5.
And here you just need to make sure that you add everything into center of the column and you'll incubate at room temperature for one minute. So it'll spin for one minute again and I'll add another 30 micro zero buffer I incubate or bedroom. So I remove the column and we have total volume of 60 micros or CDA.
Once we clean up the CDNA, you can, you can store it minus 20 degrees out and we can also dry it and then store it so we can dry the CDNA in feedback. So now in the tube we have dry CDA, which is am modified. And what we'll do is I'll suspend the dry sample in.
I'll suspend the sample in 10 ture of 15 LAR bicarbonate at pH nine. And I'll just do this by ing up and down at this step, it's very important to Resus the cDNA. Well for buying corporation reaction to work, this reaction should take place in the dark room because fluorescent labels are die sensitive.
However, the opting these from life sciences and they're, they came in individual packages. S five has purple cap and side three has orange cap and also S five appears Sion when it's getting suspended and Cary appears magenta. So what I will do in dark room is I'll transfer these samples into dye tubes, I'll re suspend the dye and transfer them back into the tube I have.
And then I'll incubate in the dark for two hours after two hours incubation these stops reaction by adding 35 re of a hundred millimolar sodium acetate at pH 5.2. And after that we cleaned up with the similar cleanup procedure. However, this time we use Cajun provided wash buffer and elution buffer.
You can describe off the places that you put the sample in and that should clean the instrument. So you can your sample or your blend right now I To, So this is my scifi labeled sample and here what we see is OD two 60, which shows the CDA content and OD six 50, which shows the D incorporation for scifi. And here is my side three label sample.
Again, twist six concentration and five 50 measures site three incorporation. Since my D corporation work efficiently, I'll combine samples Now, Ms.Miss it briefly. And now we'll write a sample again using feedback.
Okay, so for ization of the slide, we'll rehydrate by universally putting the slide on top of water and that will make spots larger. And then to protect that, we'll just snap dry on a hundred degrees Celsius and we'll then UV hybridize or UV cross link the spots into the slide and then we'll incubate in the pre high buffer that we have. So I place the slide face down, but it doesn't touch the water yet.
And we'll wait like this for five minutes. I, So this box looks larger, they should be shinier than before you put, and you'll just snap dry by putting the slide on a hundred degrees and you'll observe for condensation. And we will cross link in with youi.
So for hydration, I'll just dip the slide into pre-war water. And this should be really quick because the aim here that you want to get rid of all the spots or all the oligos on the spot that is not linked. And if you go slow in this process, you'll just get com tails on your spots.
So just make sure that you're really doing it fast and make sure that you don't take Your flight out the water and do This Stuff For 30 seconds and just fut immediately at room temperature for five minutes and 700 air pm Get This, we fit this slide into the prewar pipe station buffer that we had And slowly pushing the slide into The jar and we'll incubate this at 42 degree for at least 45 minutes. So here we have our dried sample to be hybridized. So we'll first Resus suspend in water.
At this step we just resus suspend by happening up and down and possible don't expose the sample too white, white. And then you'll add someone's sperm DNA 1.5 and the concentration is 10 milligram per mil. And you'll add 1.2 marketer of TRNA.
Concentration is 12.5 milligram per, we add tRNA and som sperm DNA to block nonspecific hydro. And we add 5.35 marker zero three XSSC, which gives final concentration of three x. And finally we add 3.5 microliter of 1%SDS.
Now we will boil the mixture for five minutes and we'll spin at the highest speed for five minutes and it'll be ready to have that. It's been an hour since we incubate our slide and right now I have water and isopropanol. So I'll agitate the slide in water first to get rid of SDS and then wash your dza propanol and directly put into the standard gauge.
And in here, just be careful that you don't expose light too much to air to prevent drying. So I go directly from harm station buffer into the water And we wash the slide. Couple minutes, just make sure that the motion is only up and down, not sideways.
And also make sure that the slide is not above water and we will transfer it into isopropanol immediately. And it just stay here for a couple minutes again. And could your page Please answer your page at room temperature for five minutes?
700 R.Cool. So our slide is also ready to be hybridized and it's better if you keep it in a slide box to protect it from the bus. So for harvest station we'll use lift or slip.
There are white strips on the cover slip and they'll provide some lift and they'll provide some space for our sample to go in between cover slip and the slide. And I clean it with ethanol And then wire put down And just make sure that there is no test particle. So first we'll take our template slide that we have print area marked, and on top of it we'll put the slide that we prepared and make sure they align perfectly.
Make sure they align perfectly. And you need to make sure that the strips are on the side, that we are gonna face it down. So it's really hard to see, but make sure that you can see it.
And the stripes should be on the sides of the slides. And we'll load our sample from the sides. I take small amounts of sample, so I take 15 microliter first and I start loading from the corner off the cover slip.
And slowly I would pull it in and I just move along the edge to cover all the area. At this stage it's very important not to introduce any bubbles and I take another 15 migrator and apply it to side of the slides to prevent any drying. So our sample is ready and you'll incubate our slides and prioritization chambers, which has micro channels to hydrate the slide while it's hybridizing.
And we'll add 10 marketer of three XSSC into six corners of the slide. There were six places on the, on the chamber, sorry, on each corner I, and on each side, Make sure that you transfer the slide really gently so that the cover slip doesn't slip away. And when you put the slide right side up, you'll see that it'll stack, it'll stick to the water that you put and put the cover And Make sure the screws are tight.
We'll incubate the conversation chamber at 65 degree water bag, but we don't wanna put it directly at the bottom. So we just put some sort of plastic barrier to prevent excess heat transfer. And we place our ization chamber gently and to prevent any displacement, you can also put some sort of weight.
And this incubation will go overnight, typically between 10 to 12 hours. So for washing of slice, we use one XSSC with 0.05 per cent SDS. This is gonna be for removing the cover slip.
And this is gonna be for washing the first step. And we'll do two more steps only with 0.06%six XSC through all the SDS that we have. So we all screw the conversation and we take slides and put it face down into purse container.
And we'll just shake it side to side and we'll see that cover slip will fall off gently. All right, and, And we'll just transfer into other wash buffer that contains XES. You aate for one minute.
Move into going oh six XSSC. And just to make sure we got rid of all the FDS, we'll repeat it one more time. Centrifuge to dry was at room temperature for five minutes at 700 RP.You keep this slide in the slide box because it's light sensitive.
So This is slide scanner and you'll put slide in here, little space right here, put the slide face down, labeled toward and, And in the second slide you'll just define the area that you wanna scan. And you'll just take this area in our slides. We have, we use 16 pins to print our slides.
So each block is corresponding to one pin and our slides contain over 8, 000 spots. And we represent vi color genome twice because it's around 4, 000 genes. So we have two spots per each oligo that we use.
And if these zoom in, you would see in this particular experiment, we compare transcription regulator mutants to wild type, and mutant would be labeled with scifi, which gives red color. And wild type was site three, which gives green color. So anything that is, that seems red, like these spots, indicates that abundance of the transcripts in the mutant was higher than the wild type.
So they appear red or shades of red, and anything that would appear green indicates that abundance of the transcript was higher in the wild type compared to mutant. And any spot that appears yellow indicates that, that that transcript was equally present in both mutant and the wild type. So when they overlay, they would appear yellow like these buts.
