 We have discussed there are many ways of printing features on the microarray based platforms especially self-free expression based systems could be very powerful to generate proteins on the chip. In this slide Napa was one of the powerful technologies which we discussed with Dr. Josh LeBair. Additionally, there is advent of many innovative ways of producing proteins on the chip without need to express and purify them. And in collaboration with University of Washington with Dr. Pradeep Rathore and his group we have tried to utilize one of the self-free expression based platform which is based on the weed germ extracts. And one of the PhD student in the lab, Apurva Venkatesh she performed a malaria based project using this novel protein microarray based technology. So today we are going to talk about applications of protein microarrays especially how it can be used for research on malaria using a high throughput microarray based platform which is also based on the self-free expression. These experiments were conducted in our proteomics lab and Apurva is going to give you some insight about how to perform such experiment and interpret data to make it much more meaningful insights from these kind of high throughput experiments. So let us welcome Apurva for this lecture on microarray technologies and its application in malaria research. I am Apurva Venkatesh for this course and in today's lecture and in the next lecture we are going to speak about microarray technology and one of its applications before we can we go on to what we can do we are going to see how to perform the particular experiment and in today's class I am going to take you to the lab and I am going to show you step wise how the experiment is performed in a laboratory. So let us move on to the proteomics laboratory at IIT Bombay. So in today's lecture I will be showing you a microarray experiment to study antibody levels of malaria positive patients to parasite antigens. For this particular experiment today first I will show you how to set up the experiment how to assemble the slides and then I will walk you through the various steps one by one. We will first begin by assembling the slides in the slide cassette. So before which I would first like to show you how our slide looks. So it is very important to know that all the slides have to be stored in a light proof box normally slides are stored in a desiccator. I have a light proof box here and I have my slides in this box I am going to first show you one of the slides and I am going to close the box. So this is basically how one microarray slide looks. If you notice this slide there are eight subarrays in the slide so let us call this subarray one and there are eight subarrays like this in this particular slide. What is important to know here is that the proteins printed on this slide are basically parasite proteins. So plasmodium falciparum and plasmodium vivax are two malaria parasites that cause malaria in humans of course there are others as well but these are the two most dominant parasites. So in this particular chip we have both plasmodium falciparum and plasmodium vivax proteins which are printed on this chip. Now how these proteins are printed I will talk to you in the next lecture. Today we will only speak about how this particular experiment is done in the lab. So what is important here to know is that each of these subarrays can probe one patient serum which means that at the same time I can study the responses of eight patients using one slide. So that is the advantage of this particular setup. So the most important thing in this particular experiment is the slide setup because if the slide is not set properly then there could be leakage and any small error in a microarray experiment can actually cause erroneous results at the end which will be very difficult for us to interpret. The first thing I am going to do is to show you how to set the slide in the slide holder. What I have here is basically the slide separator which is going to separate each and every subarray which I am going to now call pad. I am going to call one subarray as one pad. This slide holder has to be placed really carefully on the slide edge to edge. It is important to not apply too much pressure in the center or in the corners. You have to apply equal pressure throughout. This is now attached to the slide. Make sure that this is set properly and it is firm and tight. So once this is done we will now clamp this up using these clamps. We have now successfully clamped this slide. Now what we need to do is place it in the slide holder. So this is the slide holder and you will see here that this slide holder can now take 3 slides which means that if one slide can probe 8 patient sira and this slide holder can take 3 slides which means that one shot 24 patient sira can be screened. So today I am going to take only one of these slides and I am going to place it now in the holder. So this is how we place it in the holder and I will close the holder. So this completes our slide setup. The next thing we need to do is to set our reagents. So first of all what all do we need for the microarray experiment. First thing we need is a blocking buffer and the second thing we need is an E. coli lysate. So this is an E. coli lysate which is in the powder form and then we have blocking buffer here which I will use throughout the experiment. So I need to make 10% of this E. coli lysate in blocking buffer. So what I will do first is I am going to take 1 ml of blocking buffer and add it to my E. coli which is in the powder form. So I am adding 1 ml of the blocking buffer into the E. coli lysate and what I am going to do is I am going to mix it really well. This E. coli lysate which is 1 ml I am going to add it to 9 ml of blocking buffer. So this is going to give me 10% E. coli lysate in blocking buffer. So this step is basically required for incubating our serum samples before we start the hybridization to remove any anti E. coli nonspecific antibodies from the serum. So now this is my 10% E. coli lysate and I am going to place it back in ice. So once you have positioned the chamber onto the slide and the slide into the slide holder we are going to take one next blocking buffer and we are going to add this onto the chip and we are going to incubate this chip for 30 minutes. Preferably it is better to carry out this using a multi channel pipette but now that we are going to have only one slide I am going to use a normal pipette to add 200 microlitre of blocking buffer into the slide. So what I normally do is I add blocking buffer for an extra 30 seconds in alternate pads. This is basically to check if this setup is fine and if there is any sort of leakage and I am going to place it on the rocker. So when I check this I see that there is no leakage. So now what I will do is I will add the blocking buffer in the other pads. It is important to make sure that there are no bubbles in this process. So once this is done we will now carefully place this back on the rocker for 30 minutes. So while we are rehydrating our slide with the blocking buffer meanwhile what we will do we will incubate our serum samples with the 10% E.coli which we had prepared previously. So these are my serum samples which are in cryo vials. We need to make a dilution of 1 is to 100 which means that I will take 99 microlitre of the E.coli lysate in blocking buffer and I am going to take 1 microlitre of serum. So now because each pad can take 200 microlitre volume what we will do is we will take 2 microlitre serum in 198 microlitre of 10% E.coli lysate. I have already aliquoted the 10% E.coli lysate. Now what I will do is add 2 microlitre of serum samples into this. So usually this is also done using a multi-channel pipette or sometimes there are also automated ways to do this. But since we are again dealing with only one chip I am using a normal pipette. So that way we have now incubated 2 microlitre of 8 serum samples in E.coli lysate and we will incubate this for 30 minutes in the same platform rocker. So now it is time to add our serum samples to the slide. So the first thing we need to do is to aspirate the blocking buffer which is already present on our slide. For this I will take the multi-channel pipette. So this step has to be performed really carefully. As you will see these are really fine tips which have to be used to avoid any scratches. It is important to keep the pipette at the very end of the slide this way and then you aspirate really slowly. The other important thing is that the slide should never dry up so it is always good to keep a little blocking buffer behind. As you will see the blocking buffer has been completely removed and now immediately we will add our samples. So this is the 50 microlitre pipette. I am going to mix the serum sample once before adding it to the slide. So once this is done we will add the serum samples to the slide. So we have totally added 200 microlitre of serum samples which have been diluted in 10% of E.coli lysate. So to avoid any kind of evaporation what we will do is we will place a foil over the lid and then we place this whole slide on a moist towel. We will then place this slide overnight in a cold room which is at 4 degrees. So after overnight incubation the next thing we need to do is aspirate this solution just like we did previously and we are going to wash this pad with tween buffer. I am not going to show the washing steps now because it is similar to how we aspirate it. The same way you aspirate and then you wash it and then you aspirate again. This has to be done thrice. This is our secondary antibody. What we will do is we will dilute this 200 times. I am going to take one microlitre of the secondary antibody in 199 microlitre of buffer and I will calculate this for 8 pads. So basically I have already calculated the volume required for 8 pads and I am going to simply add 200 microlitre of secondary antibody to each pad. It is important to not scratch the pad and also as I have mentioned earlier the pad should never dry. So once this step is done we will again incubate this slide on a platform rocker. Okay so now what we have done is we have incubated this slide with the secondary antibody and now we will incubate it with the tertiary antibody. This process is again done after washing the slide thrice with tween buffer. The dilution for this antibody is similar to the previous one, 1 in 200 and I have again calculated accordingly. So now we will add 200 microlitre of tertiary antibody which is a strepta with ridden conjugate to the slide. It is very important to close this slide and wrap it with foil immediately as this tertiary antibody is light sensitive. So once this is completely wrapped we will again place this on a platform rocker. I would like to mention that after this step every other step has to be performed very carefully and we should try to avoid light as much as possible. So after incubation with tertiary antibody we are going to now wash the slides just like previously with tween buffer. So every time you add tween buffer you place it on a high speed rocker for 5 minutes and then you wash it again. So this process is done 6 times. Now it's time to remove the slide from the slide chamber for scanning. So I will carefully remove the slide from the chamber, I am removing the clamps. You have to be careful not to scratch the pad surface. So we slowly remove the chamber from the slide and then we immediately transfer the slide into the slide holder. So this is basically distilled water to wash the slide before we scan it. So we have a slide holder with the slide and a balance which we will now centrifuge at 2000 rpm for 5 minutes. So we are going to now scan the slide as you will see the slide is dry after centrifugation. We will place the slide carefully in the scanner with the barcode side facing downward. So now we will scan the slide. So hope you are familiar with how to perform the microarray experiment. So it is not difficult, it is just a little tedious and there are certain precautions which you need to take while performing this particular experiment. So basically it is very important to store the slides in a light proof box and in a desiccated cabinet if you have it. QC of each sample has already been done I am sure but it is important to ensure that it has been done because these are all IVTT spots on the chip and they have to be checked once before you receive them. But it is important to probe one no-seria control in every batch I have already shown you that but it is very important in probably in every slide if you can probe one it is great. If not at least in every batch there has to be one no-seria control which is nothing but your blocking buffer plus 10% E. coli lysate. The pads must never ever go dry otherwise you will actually see huge background noise which is very difficult to then later on eliminate. So you have to use autoclave tips all the time and the last thing is to label the slides very carefully otherwise towards the end of the experiment you will never know which slide was used for probing which samples and especially when you have a large number of samples like 200 and 300 it becomes very confusing at the end. In the next class I will teach you how to analyze data using Excel I will show you how to what you do after you export data from your microarray scanner. This will be very helpful for a few of you who are just starting a microarray experiment in the laboratory. See you next week. Thank you. Now you are convinced there are many ways of producing the contents on the chip in different ways both cell based or cell-free expression based manner. Cell-free expression provides lot more flexibility that you can generate large number of proteins of interest without need to purify them. And especially in this case when we want to do a research on malaria we had different pathogens different parasites from which these genes were the clones were made from felsiparum and vivax and they were printed on the chip and thought was can we express them using in vitro transcription transition mix and made the protein on the chip and then use those to screen the patient's biological sample. I hope you got some understanding about how to perform a microarray experiment in our proteomics lab which was demonstrated today. You also understood every step is so crucial in high throughput biology if you are looking at every step meticulously your protocols your SOPs your quality control checks are in place then only you can obtain a result which could be reproducible and meaningful. In next lecture we will continue our discussion about using such approach and application and how to then analyze the data and make more meaningful insights from this kind of experiments. Thank you very much.