hi subscribers soon to be subscribers and lurkers welcome to this session on
analyzing microarrays when you hear the term microarrays you should know this: they may be
dna microarrays or it may be referring to protein microarrays for dna microarray the
sample you start with are messenger RNA (mRNA) that have been reversed
transcribed into dna (cDNA) that you can use them as substrate on your micro dna microarray chip
now the arrays itself are just solid supports usually glass or it could be silicone upon which
you've either got gene specific probes if you're doing d if you're doing dna microarray or you may
just have antibodies if you're doing protein microarray. now within the protein array it
may be antibodies that are embedded on the glass surface on each of the spots on the
microarray slide or it may be purified proteins or peptides so you can have acids that are looking
at functional proteins or protein interactions or you may just be looking at the presence of a
particular protein and in this case you just have to have an antibody embedded on the glass slide
protein microarrays allow you a more high throughput assessment of your proteins via
antibody and because you're using a microarray slide you essentially will need a micro array
reader and the kind of data output then would be a lot more than if you had essayed it
using elisa and so in this video we're going to go over how you analyze your microarray data
and so the way that the slides are read is typically for in a zigzag pattern so each block
it's arranged if it's a 16 block arrangement then slide one is on your left the top left
and slide and and block one sorry it's on your top left and block two is on your top
right and then three will be on the next block on the left and then so it's in a zigzag
side by side pattern and that's how the reporting from the scanner will be given to you that's how
you will assess it and so you have your standards at the top of the slide in my particular essay i
had eight standards so the top eight blocks were my standards where the highest concentration was
block one the next concentration was block two and then the serial serially diluted concentrations
just followed and took up eight blocks which meant i could put eight samples on my 16 block slide
and then on your subsequent slice if you're doing multiple slides in a day you could
just pick one of the standards as as a quality control as a quality control to place on your
next slide and you can also have a specific quality control sample that previously i said
and know the amount of on the side as well so you don't have to take up the whole eight
blocks as standard again if you're doing draw in the same day that is and so on
and so forth for the additional slide so include one of your standard curves maybe
one of the standards used for the standard curve maybe the middle one and then include a quality
control sample so a sample you've already measured and know the amount of so you know what you're
expecting just so you can track if anything goes wrong in the subsequent slides that do
not have the standards on it now as mentioned when you go to scan you will have the cytokines
or whatever you're measuring varying greatly in the same array because you've got different
cytokines or proteins that you're measuring at different
different amounts being expressed and so you would have to change the photo multiplier tube
to be able to gauge for low signals so you use a higher photomultiplier too for low signals and a
low photomultiplier tube (PMT) for high signals and so you may need to take multiple scans
once you have your scan what you need to do is align a file which tells the software
which where the blocks are that it needs to quantify so this is the gal file that you will
align onto the acquired image each slide that you purchase will have a guile file that matches the
arrangement of the arrays on the slide or if there isn't one and you've created it yourself
then you have to create your gene alignment file so once you've aligned it then depending
on your software you can go ahead and get the extracted values for a quantify the value so
in this example i'm showing it for the software mapix and so with mapix you go ahead and do a
preview scan and then once you have the preview scan you have the opportunity to adjust it for the
type of scan you want to do whether you want to do an automatic focus scan and manual and well
where you tell the software what intensities you're looking for or you can do an extended
dynamic range scan so that you can capture both the low it can be optimized for both the low and
high but you know it's gonna it's not going to be ideal so you probably need multiple scans
in determining the pmt setting the aim here is to avoid saturated pixels depending on your software
the saturated pixels may show up as white blobs which would indicate to you that you're saturated
and so you need to dial down the pmt so that you can accurately quantify that signal that block
so these gal files aims in in describing the size and position of the blocks if your assay does not
have an associated gal file because you didn't purchase it from a manufacturer then apparently
you can create one in excel as long as you export it as a tab delimited format once you have the...
once you have the correct alignment and you've determined that you don't have any saturated
pixels the right image has been scanned, you can go ahead and select in your software the icon
that allows you to quantify. and in my case, in the Mapix software, the icon is the 'photometric
calculations'. When I do that i get a long list of quantified values that i can now
go ahead and extract those values and analyze using an appropriate macro
excel setup macro that will identify what the standards are and ma and plot it and using
either a non-linear or a log-log regression fit i will be able to get... calculate the unknown
concentrations for the samples that i want to determine the amount of protein that i have
and so it's really handy if you have a macro and excel setup macro that you can use now you want to
normalize the quantified values that you've obtained the fluorescent intensities from one
within the same slide may change depending on the block that you are assessing so there are positive
control and negative values on each block that will be used to normalize for each of the other
blocks so you know what intensity a positive should be for that particular cytokine and what
a negative should be and that will be factored in to normalize all the other blocks if you had
tens if you had hundreds if you had thousands each one will have its control and that's what
we call intro slide normalization then it off for normalizing if your essay ran over multiple slides
so if your sound because you have so many samples that didn't all fit on one slide fluorescent
intensities from one side will vary can vary from one slide to the next so once again we do an inter
slide or between slide normalization and here is where your qc sample your quality control sample
or the standard reference that you repeated on the multiple slides can be used as a tool for gauging
how the intensities have risen or dropped from one slide to the next it's only after you've corrected
and adjusted for these intensity variations that can occur with the experiment that you go ahead
and plot your standard curve and interpolate read of what the unknowns will be and for that
you can use any software that allows you to do this you can use excel you can use the
arrigo elisa data calculator that i've shown in the previous video i'll have it linked here
so hey at this point how about giving me a thumbs up and subscribing if you haven't subscribed if
you're finding this useful thank you i appreciate it okay so now you're going to go ahead and plot
and get your unknown concentrations and then there's a further step for your data analysis
now the final step would then be to use a graphical software in my case i just use Excel
everyone has excel but if you have a dedicated graphical software then you're just going to go
and plot the values that you've recorded based on the values that you've recorded for the different
conditions or groups or experimental setups that you have and see which how the conditions have
affected the reported value for that particular protein that you measured whether it's a cytokine
or something else if you're looking for help on how to plot this then i have a link to my Excel
videos and how you do the plotting there it like i said excel is a great place to start because
you know everywhere you go you do have access to that so once you kind of master how to do
it in excel you are always able to present to your data graphically when you need to
normalisation is removing variations that are due to the technique rather than the biology
of the samples or the samples you're you're studying normalisation is removing technical
variation so that you can have biological insights that are genuine