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Running ELISA, Western blots and RT-qPCR with the Case It molecular biology simulation — Transcript

by Case It molecular biology simulations · 2,983 words · 464 segments · language en · Watch on YouTube

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0:00This tutorial has been updated to
0:02reflect modern methods for HIV
0:04detection. Beginning with a
0:06demonstration of the ELISA capabilities
0:08of case version 705.
0:12The first thing we need to do is to
0:14click on the protein button on the
0:15silver button bar which is located on
0:17the left
0:21and then here is the open dialogue box.
0:25Locate the cases folder. Then double
0:27click on it.
0:29locate the infectious disease folder and
0:32we will use HIV as an example and then
0:36the example I would like to use is
0:38located in the US folder. So I'll double
0:40click on that and ANA is the case that
0:43I'm interested in right now for
0:45demonstration purposes.
0:48Inside the Anna folder are three folders
0:50and the one of interest right now is the
0:52one that includes sequences for the
0:54ELISA and Western. And since I had
0:56originally clicked on the protein
0:58button, that's what I want to select. I
1:00could either click that and click open
1:01or I could doubleclick. But in this
1:03case, I'll just click open. And you'll
1:06notice that that protein file is now
1:08open. It appears in the open and process
1:11window.
1:12It contains characters in six separate
1:15sequences. And we can see those
1:17sequences if you clicked the sequence
1:19button. You don't actually have to do
1:21this to run the ELISA, but just for
1:23demonstration purposes. Here are the HIV
1:26protein sequences
1:29that we will be working with.
1:33The next thing we need to do to run an
1:35ELISA is to open up the patient samples
1:39that may or may not contain antibodies
1:41to HIV. So I will click on the antibbody
1:44button on the silver button bar
1:48and then whatever one happens to be at
1:51the top in this case it's trimester 1. I
1:54will click on that to highlight it. I'll
1:56move down to the one just above the
1:58protein. In this case you just want to
2:00select all those antibbody sequences and
2:02click open.
2:04And they will appear in the open and
2:06process window.
2:08So now what you should do is click on
2:11the protein to make sure that that is
2:14activated and that's what will line the
2:16wells of the ELISA. As an aside, if this
2:19was a fourth generation ELISA, the wells
2:22would also be coded with antibodies to
2:24the P24 HIV capsid protein, making it
2:28considerably more accurate than earlier
2:30generations of ELISA.
2:34Then you select the first antibbody
2:36sample, whatever it happens to be. In
2:38this case, it's Anna trimester 1. Hold
2:41down the shift key and select the last
2:43antibbody sample. Highlight all of those
2:47and then use the quick load and run
2:49button and go down to elysa and option
2:52one load the same protein and different
2:54antibodies. There are other cases that
2:56might where option two would be
2:58appropriate, but in this case, option
3:00one is appropriate. So that's what I
3:02will select.
3:04This is a quick load procedure, so it
3:06automatically loads the ELISA plate, but
3:08there's a way of doing this step-wise as
3:10well that I'm not going to demonstrate
3:12in this tutorial. So, now that it's
3:14loaded, all we have to do is click the
3:16run button here to run the ELISA.
3:21And you'll notice that the wells were
3:24automatically labeled when you use this
3:26procedure. And to make this a little
3:28more realistic, you normally would read
3:29an ELISA plane on a light box.
3:34Go to the options button.
3:36Click that.
3:39Select ELISA. And what we will use in
3:41this particular case are absorbance
3:43values, optical density. So I will
3:46select that. And you'll notice that now
3:48these optical density values have
3:50appeared. I'm not going to try to
3:52interpret this right now, but I would
3:54say that for something to be considered
3:57positive, it should be at least twice
3:58the negative control. And here you can
4:00see the the one that's not very clearcut
4:02is the boyfriend. It's fairly faint and
4:05it's just about twice the negative
4:07control. So we're a little unsure of
4:09that. And remember the ELISA is just the
4:11first test you would do if somebody came
4:13in to be tested for the presence of HIV.
4:21What we really need to do if we're
4:23unsure of our results here is to run a
4:25more definitive test which is the
4:27western blot.
4:39And now I can very quickly run a western
4:42blot. Again I will demonstrate the
4:43autoload features.
4:46Go ahead and recclick on protein. You
4:48always want to redo this clicking before
4:50each procedure. So click on the protein
4:53to activate it.
4:55do a shiftclick to include all of those
4:58antibbody samples
5:00and then go to quick load and run
5:03and down to western blot and just like
5:06for the antibbody we want to load the
5:07same protein and different antibodies.
5:11So I will select that it will
5:13automatically load the wells and now I
5:17could go to the click load and run
5:20button down to western blot and I can go
5:23ahead and run it after loading the
5:24samples. Now I could have just selected
5:26this option to do it all in one step but
5:29I didn't. I selected this option and
5:31this option. So now we have to actually
5:32run the western blot. So I will select
5:34that.
5:36Unlike the eliza the blot is not
5:39automatically labeled. So I can quickly
5:42do that by highlighting
5:46one antibbody trimester
5:51copy into the gel label. Two antibbody
5:54trimester 2 copy
5:58three antibbody baby
6:01four antibbody boyfriend
6:07five the negative control.
6:16Click to the left of the two and space
6:18that. Click to the left of the three and
6:21space that.
6:23Click to the left of the four. Hit the
6:25enter key. Click to the left of the five
6:27and then space that over. And then do
6:29the same thing for six.
6:32The elysa plate has been lined up with
6:35the western blot. So we can compare the
6:37results.
6:39Keep in mind this is a historical
6:41example from a number of years ago when
6:43western blotss were considered the gold
6:45standard for HIV detection. That's no
6:49longer the case. There are other
6:51procedures used, one of which will be
6:54demonstrated later.
6:57Just use your imagination, put yourself
6:59back in time, and this is the way things
7:01would have been interpreted a number of
7:03years ago, especially towards the
7:05beginning of the AIDS epidemic.
7:08And it does show the value of western
7:10blotss which is still a very commonly
7:13used research technique for other
7:14purposes.
7:16Anna in trimester one in this example
7:20had a relatively low OD value indicating
7:23that she was negative for HIV during
7:26trimester 1. And you can verify that
7:29looking at the western blot. There's no
7:31antigen antibbody reactions here. No
7:34bands are showing in lane one. In
7:37trimester two though, she had a higher
7:40OD value with a strong positive result
7:43for the ELISA. All six bands were
7:46showing for the western. And the
7:49criteria back then was that bands two,
7:52four, and six were showing that was
7:55considered definitive. So back then she
7:57would have definitely been considered
7:59definitive for HIV. Her baby had a
8:03strongly positive elysa,
8:06but notice only two of the six bands are
8:08showing in lane three of the western
8:11blot and it doesn't match the CDC's
8:15criteria back then. So the baby would
8:16have been considered indeterminant and
8:19additional testing would have been done
8:22for lane four for the western. All six
8:26bands are showing. And that's
8:27interesting because that's the
8:28boyfriend's
8:30result. And if you look at the ELISA,
8:33notice an OD of 222. Compare that to the
8:37negative control of 1.02.
8:40So that's just a little bit over twice
8:42the negative control. So that's right on
8:44the borderline for the ELISA. But for
8:47the western blot, which was considered
8:49definitive, that indicates that he
8:51definitely had an HIV infection. And
8:55then for lane five of the western is
8:58negative, which you would expect because
9:00that's the negative control. Lane six is
9:03positive, which you would also expect
9:05because that's the positive control.
9:10And now we're going to come back to the
9:12present and look at one of the
9:13techniques that's used for a more
9:16precise HIV diagnosis. That technique is
9:19abbreviated RT qPCR. And in this
9:23technique, viral RNA from a patient's
9:25blood sample is reverse transcribed into
9:29cDNA and is put into a specialized
9:32thermosycler so that the resulting cDNA
9:35can be quantified. So we'll use the DNA
9:39button
9:42to open samples. Go to the cases folder,
9:45the infectious disease cases,
9:48HIV US ANA. And now we'll go to the
9:53viral load folder. And we will be
9:56opening three DNA files. These would
10:00actually be cDNA that had been reverse
10:04transcribed from viral RNA. So these are
10:07the three samples. So that first step is
10:10not shown. We're just starting with the
10:11DNA itself for this example.
10:16Since it's PCR,
10:18we need to open a primer
10:24and then simply shiftclick the three
10:27Anna samples from 1 month, 3 months, and
10:306 months because we're trying to follow
10:32her viral load over time, assuming she's
10:35under treatment. Go to the quick load
10:37run button down to 96 well PCR. This is
10:41really 96 well qPCR.
10:44Load the primer and the DNA samples
10:48and then click the run button.
10:52Now, you really wouldn't be seeing this
10:54result inside the machine, but this
10:56would indicate a certain point in time
11:00on the thermosycler
11:02showing that in
11:05one month's time, Anna had a fairly
11:07substantial viral load apparently from
11:10this positive result and then it seems
11:12to fade over time. To see what the
11:15actual numbers are, go to the options
11:17button down to 96 well PCR and viral
11:21load.
11:25The results indicate that her therapy is
11:28quite effective because the numbers go
11:31from 55,000 units to 5,000 down to 150.
11:38We're going to demonstrate a more
11:40realistic depiction of the qPCR process
11:43using that feature of Case. The data
11:46that we we will be using is accessed via
11:50the qPCR button on the silver button
11:52bar. Open file containing qPCR
11:55fluoresence chart data
12:00and the data is called qPCR honeybee.
12:04That's because this data came from an
12:06actual research project at the
12:08University of Wisconsin River Falls.
12:10It's very difficult to get a hold of
12:12fluorescent data for HIV patients. So we
12:15will use this as a surrogate data set
12:18and we'll only be using part of the data
12:20set.
12:22So I click the open button to open the
12:24data. We don't need these windows open.
12:28And I'm going to move the chart up so
12:29that it's visible on your screen. Next,
12:32click the load button right here.
12:37And that will load all 96 of the wells.
12:41Go back to the qPCR button and select
12:43run qPCR for orange wells only.
12:52So this is what the qPCR software would
12:55generate for an actual machine. You end
12:58up with these curves that start out
13:01growing very slowly, then grow
13:03exponentially and then level off like a
13:05logistic growth curve.
13:08That's because with each step of the
13:11thermosycler, the DNA is being
13:13amplified. It's a series of heating and
13:15coolings. So not until 20 or 22 does it
13:19do you get enough DNA where starts
13:22growing exponentially and then it will
13:24level off.
13:27Where each one of these curves
13:29intersects this green line which is
13:31called the threshold.
13:33That number here is the CT value. And if
13:38I'm going to just move my cursor over
13:41some of these,
13:44but so well C6, for example, has a CT
13:47value of 24.47. In other words, about 24
13:511.5 cycles until there was enough cDNA
13:54in that sample to hit that threshold. So
13:58that's well C6 24.47.
14:02If we look at well C6
14:05right here 24.47.
14:09So that's where those numbers come from.
14:11It's called the CT value.
14:13And the lower the CT value, the more
14:16cDNA there was in that sample. That
14:19means that it will start that
14:20exponential growth phase earlier because
14:22there was more cDNA to start with. The
14:26bigger the CT value, the less cDNA there
14:29was in the original sample.
14:34We'll now use the honeybee data set as a
14:36surrogate data set for HIV samples.
14:40Assume for the sake of argument that
14:43well C6 is one of Anna's samples from
14:48the HIV case. And let's further assume
14:51that was one of the samples that was
14:53done at 3 months.
14:57See what would happen if we just ran
14:59qPCR for that one sample. We'll go to
15:01the button run qPCR for purple wells
15:05only and it gives a value of 24.47 as we
15:09showed before but now you can see this
15:12single curve and you can see where it
15:14crosses the threshold. If you hold your
15:17cursor it says 24.53 that's because it
15:20depends on which pixel that cursor hits
15:24but the actual value is 24.47. So this
15:27value will be close to that value.
15:30Normally these are run in triplicate. So
15:32we will select two more wells
15:36back to the qPCR button and run that for
15:39purple wells only. And now you can see
15:42that they are pretty tightly clustered
15:45together. So we will use that average
15:47value a little bit later of those three
15:50CT values.
15:53So again that was Anna's samples at 3
15:56months. Let's do three samples at one
16:01month. And for that, I'm going to select
16:04E4, E5, and E6. As you can see over
16:08here,
16:11let's go ahead and run that for purple
16:13wells only.
16:18These are more to the left indicating
16:21that there must have been more viral DNA
16:23in our samples at that point in time
16:25because it crossed the threshold earlier
16:28than the sample at 3 months. So these
16:31again would be the three triplicates
16:32from 1 month with a value somewhere
16:35around 22.6 or so. These are the three
16:39actual values. We'll average those and
16:41use those average values later when we
16:44look at a standard curve.
16:46And finally, we need to do her samples
16:48at six months. So, we'll select
16:53B7, B8, and B9.
16:57Go to the qPCR button and say purple
17:00wells only.
17:03And now we have all three sets of data.
17:06Notice these are not as tightly
17:08clustered together, but all three of
17:10them are more to the right of this one
17:13and this one, indicating that there was
17:15less viral RNA in her sample at 6
17:18months. To determine viral load from
17:21this data, we need to generate a
17:23standard curve. First thing I will do is
17:27go to the clear menu and say clear
17:30everything. Bring this one up.
17:34Select the qPCR button. Open file
17:39and this time select the standard curve
17:42data set.
17:45Click the load button.
17:48Run qPCR for orange wells only.
17:54This standard curve was run with
17:56duplicates.
17:58So you'll notice that they are
18:00consistent with one exception and it's
18:04this one right here.
18:06So these are very close
18:10as are these.
18:12This looks like an outlier
18:15and notice it is well D2.
18:23Looking at the amplification plot here,
18:30this is the line for well D1 and D2 is
18:32way over here. So that's clearly an
18:35outlier. Something was wrong with the
18:37experimental procedure. If you look at
18:39the standard curve, which is going to
18:41plot the [snorts] initial quantity
18:44copies versus the CT,
18:48you can see that this is clearly an
18:50outlier. to be very careful about
18:53outliers because sometimes they can be
18:54the most important data point in a set.
18:58But in this case, it's clearly something
19:00on this with the experimental procedure.
19:02So, we're going to rerun that for all of
19:04the wells except for well D2, which is
19:07the outlier.
19:09To do that, first I need to go back to
19:11the amplification plot.
19:14and before I got to the standard curve
19:15by clicking this button.
19:18But we need to be on the amplification
19:20plot. And I'm going to go to well D2 and
19:24select it to turn it purple. And then
19:29run QPCR for orange wells only.
19:34And you'll notice the outlier is no
19:36longer there.
19:39Going back to the standard curve,
19:43you can see that that data point is
19:44missing. And now what we want to do is
19:48plug in the average CT values that we
19:51generated before. The average of those
19:53three values for 1 month, 3 months, and
19:566 months into this box. To do this,
19:59we're going to need to magnify the chart
20:01because if you leave it at this size,
20:03some of these numbers are going to be
20:04cut off. So, I'm going to hit click this
20:08magnifying button.
20:11Bring this up.
20:13So, the number I'm going to put in here
20:15for Anna at 1 month using again the
20:20honeybee data as a surrogate for Anna's
20:22data, the average value of the CT for
20:26E4, E5, and E6 for those three wells was
20:3022.64.
20:32So, I'm going to type that in.
20:39And you can see the result the number of
20:42copies 1.745e
20:46plus 03. That's scientific notation and
20:49it means that the decimal point should
20:51be moved three places to the right. So
20:54that would be 1745 copies. 3 months the
20:58average of the CT values for cells C4,
21:01C5 and C6. I calculated that is 24.43
21:10giving interpolated copies of 5.149E
21:14plus 02. So move the decimal point 2
21:17units to the right that'd be 514 or if
21:21you round up 515.
21:23And then finally for on it 6 months if
21:27you recall that was cells B7, B8 and B9.
21:30The average for that I calculated is
21:3225.68.
21:40Moving that two units to the right
21:42that's 219.
21:45So at one month there were 1745
21:49copies. At 3 months 515
21:52and at 6 months 219.
21:55So the viral load is decreasing over
21:57time. Even though those are
21:59unrealistically low values for a real
22:01HIV infection, this example does show
22:04how qPCR can be used to determine viral
22:07load.

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