Hi, I'm Josh Sh, who I'm a graduate student at, at Dr.Ed Maki's lab at the University of California Irvine. I'll be showing you how to extract RNA from neuro precursor cells using this BioRad to RNA mini kit. I've been using this kit for a couple years already and it's at this lab and it's always been giving me consistent results at high yields.
So the cells that we use are Eton and a half neuro precursor cells that have been acutely dissociated from embryonic cortices, and we plate them usually in a 24 well tissue culture treated to plate that has been PD L and laminate coated, and I plate them at a hundred thousand Cells per well. Before I start, I have to tell you that you should work on a, a designated bench where you do do your RNA work. First things first is to make sure your RNA bench is clean.
Appropriate way to clean the bench is to use this RNA zap. Basically it kills off any RNAs reason being that the RA basically destroys your RNAs. Other thing is to wear gloves always when you're handling the extraction because your skin's filled with these RNAs.
So it's a big no-no. Okay, so what I have here is the BioRad total RA mini kit. I've taken out its contents, so let me show you what they are.
First thing is we have this one X dep C treated PBS, and we have 70%DEP treated ethanol. We have total RNA license solution that has Dan me capital ethanol added to it. Second is a low stringency wash that has a hundred percent ethanol added to it, the high stringency wash, the DNA dilution solution, and the DNAs itself and the total RNA elution.
So solution, which we'll put into the 70 degree heat block. The other things that we need are 1.5 mil cap tubes. These don't contain any DNAs or ais, so make sure you have tubes that don't have that stuff.
And we have two mil capless tubes. And the most important thing of all is the RNA columns itself. Without it, you can't do the RNA extractions.
So in order to assemble these columns, you need the the RNA column and you need the two mil capless two and just put it together like that and you should be ready to go. Okay, and the last thing that we always need are these cells. So the cells here are Eton and a half neural precore cells.
There were acutely dissociated from embryonic cortices. They've been grown for a couple days in temple media. I've taken the cells out out of the 37 degree incubator.
This protocol is specific to adhere cells. These cells were grown on tissue culture treated 0.4 well plates that have been PDL and laminate coated. So now I'm gonna take a vacuum hose and I'm gonna ask, break the media off.
And the next step is to add around 500 microliters of the dep SEA PBS. And again, we're, we want to use filter tips. Now, the PBS, what it does is basically it washes off the media.
So you don't want any of the media components into your RNA column or interfere your RNA extraction and you won't, you won't get good yields.Okay? Gonna quickly aspirate off the PBS because the neural stem cells, they don't like the PBS. Now I'm gonna add 350 microliters of lysis buffer to each.
Well Again, make sure you have beta my capital ethanol in the license buffer. Now I'm gonna pipette up and down the lysis buffer 15 times to make sure the the cells are lic. So we can get the RNA contents out.
Now I'm gonna add 70%deeth ethanol to each well, after I add 350 microliters of this 70%deci ethanol, I'm gonna pipet up and down each, well 10 times. I pipetted up and down 10 times for each. Well, now I'm gonna set my pipetter to over 700 microliters since the total volume of each well is 700 microliters.
Then after I pick up the stuff that's over 700 microliters, I'm gonna dispense it into a RNA column. After adding the contents of the column, I'm gonna go to the centrifuge and I'm gonna spin it for 30 seconds at full speed. After the first 32nd spin of the lysate, I'm gonna discard the filtrate by dumping it into a empty container.
Or what some other people do is they take a backing hose and aspir it off the filtrate. Next is you're gonna add 700 microliters, the low stringency wash buffer. And again, make sure you add the ethanol into it.
Now I'm gonna spin it for 30 seconds at full speed. After spinning the low stringency wash buffer, I'm gonna discard the filtrate.Okay? Then I'm gonna prepare the D, the DNA solution.
So for each sample you're gonna add five microliters of DNAs. So for us, we actually aliquot the DNAs before we ever start this. cause again, it's a enzyme.
It's better to store them as aliquots. If you store them as a whole, the freeze and thaw will destroy the enzymatic activity. So after I add the five microliters of DNAs, I'm gonna dilute it with 75 microliters of the DNA dilution solution.
So after you add that, the total volume will be 80 microliter DNA solution. Now I'm gonna add 80 microliters, the DNA solution to each sample. Make sure get it down onto the membrane of the column.
So you want that DNA solution soaking up the membrane. So you'll get rid of the DNA. So you'll increase your RNA yield.
So after you add the DNAs, you'll let the column sit for 15 minutes at room temperature so you can let the enzyme do its work. And again, you'll incubate it for 15 minutes. Alright, timer is off.
After the 15 minute incubation, I'm going to go to centrifuge and spin down the DNAs 30 seconds at full speed. Okay, after spinning for 30 seconds to get rid of the DNAs, I'm gonna discard the filtrate. Now I'm gonna add 700 microliters with a high stringency buffer to wash out any residual DNAs.
And then after I add that, I'm gonna spin again for another 30 seconds in the centrifuge. After the spin, I'm gonna get rid of the high stringency filtrate, gonna discard it by just dumping it to a container. And we're gonna add 700 microliters of the low stringency buffer.
This time, I'm gonna spin it for one minute at full speed at the Centri Beach. So after the first one minute low stringency wash, I'm gonna discard the filtrate. And this time I won't add anything, but I'm gonna spin again for another two minutes in order to get rid of the extra low string seed, because the low stringency has some ethanol into it, and that will lower your RNA yield.
Now I'm gonna get the illusion buffer out the 70 degree heat block. So just to remind you, make sure before you start the experiment, make sure you put the elu buffer is 70 degrees and we want to keep it warm so it'll increase the efficiency of your RNA yield. So I have the columns ready.
They have been taken out in the centrifuge. So next steps, they take out the columns. Okay, I'm gonna discard the cap tubes and we're gonna transfer em to these cat tubes.
You are gonna add 50 microliters of the Lucian buffer to each column. Make sure you get the illusion buffer onto the membrane so that it soaks it up really well. If you don't, you won't get good RNA yield.
Now you let the column sit for one or two minutes in order to ensure that the illusion is soaking up the membrane of the column. Okay, it's now one minute. Now I can take it to the centrifuge.
Now I'm gonna put the columns in the centrifuge. I'm gonna spin it for one minute at full speed. And this is the last centrifuge step, which you'll collect the el pollutant or the RNA into these caps tubes.
Now, after the last spin, take out your contents, remove the column, you can discard em, close the tube, and now you can store them away in the minus 80, and you can do whatever you want with them later on. So I've just extracted RNA using the BioRad RNA kit. Now I'm gonna measure the concentration of the RNA that we extracted, or we can read the OD of it using a spectrophotometer.
So this what I'm gonna show you. So first we have to make a dilution mix. So it's gonna be a one to five dilution.
We're gonna mix our RNA and just molecule MQ filtered water. So I'm gonna first make a blank. So I'm gonna grab two microliters of just our elucian solution that we use to elude our RNA early on, and I'm gonna add eight microliters of the molecule water.Okay?
So that's our blank. Now I'm gonna prepare the sample. So it'll be two microliters, our RNA of interest.
We're gonna add, again, eight microliters of the MQ water. So that'll make it a one to five dilution, right? So I have this glass ette where I'm gonna put my samples in and I'm gonna load this ette into the spectometer.
So let's go measure this. So before I measure the OD of the RNA samples, I'm gonna have to clean my vete. So again, here's the glass vet.
You never ever want to touch the front of it. So you'll leave any prints on it and I'll give you wrong measurements. So gonna, what I'm gonna do first is I'm gonna wash the glass key bet with double DI's water.
Then I'm gonna take a vacuum hose. I'm gonna suck out the water. After that wash, I'm gonna wash it with 95%ethanol, and then I'm gonna use a vacuum hose again to remove the ethanol.
Now I'm ready to load my blank. So we need the blank first in order to normalize our readings. So I'm gonna get 10 microliters of the mix that we just made.
So I'm gonna mix our solution up and down and get a nice mixture. I'm gonna bring it to this spectrum speedometer. This is the Ultra Spec 3, 100 probe machine, and we're gonna measure the RNA concentration.
So open the machine, gonna put it into one of these holders. We're gonna manipulate the programs such that while read RNA only. Okay, again, we did a one to five dilution.
So we want to set our dilution factor to five. Okay, so I'm ready to go. So I'm gonna press run in order to reference the blank.
All right, it's now zeroed out. So now we're ready to measure our sample. So I'm gonna take the glass key bed out.
I'm gonna suck out the, the contents of the blank. Again, I'm gonna wash it with double DIS water. I'm gonna wash it with nine 5%ethanol.
I now I am ready to load 10 microliters of our sample of interest. So again, make sure you want to mix your sample. Well load it into the glass vet.
Now we're ready to read. After measuring the od, this is what a good yield I parity should be an RNA absorbance ratio of two 60 to two 80, should be around two. And the yield is about 144 micrograms per mil.
Even though the machine says 72 micrograms per mil, we have to multiply this by two because of the half length in our vets, two centimeters showed you how to extract RNA from neuro precursor cells. Again, we are using the total mini RNA kit from BioRad. We tried other kits and this is the best kit that we use based on its efficiency.
That's, that gives RNA at at a high quality. If you do use this kit, you'll get the same high quality results that we always get.
