I don't understand this question... is the assay failing with analytes that are known to be present?

Yes assay is failing with the analytes that are known to be present. I am working on hybridization of probe coated on beads with biotin labelled PCR product. It was working very well showing increase in fluorescence when single PCR product and bead combination was used. However, in multiplex the fluorescence signal was same as of background containing other things (Probe, SAPE,) but no PCR product. I am not able to differentiate whether Target PCR product is there or not?

Is the background still low, or has the background risen?

Did all the target analytes work as a single plex?

Are there any differences in the sample that you are using between the single plex versus the multiplex. And how big is the multiplex, how many analytes are you trying to detect?

Background risen many times, it doesnot remain constant. sometimes its more than the positive control. Yes all the analytes are working good as singlex and even working good as multiplex even in conventional PCR. I am taking three analytes for detection and sample is same for both singlex and multiplex. It is the bacterial DNA isolated from pure culture serving as positive control.

If the background keeps going up, my first though is DNA contimination, or something else that is causing your SAPE to mark everything, or there are endogenous biotin.

What do you mean it worked in conventional PCR? You mean the probe sequences that you are using worked in a TagMan assay?

So the single plex worked with the beads? And when you mixed the beads from the single plex, it didn't work?

Q. What do you mean it worked in conventional PCR? You mean the probe sequences that you are using worked in a TagMan assay?

A. no not by taqman. it means probe sequence is showing amplification with reverse primer by conventional PCR giving the appropriate product size of 800 bp (Forward probe and Reverse biotin labelled primer).

Q. So the single plex worked with the beads? And when you mixed the beads from the single plex, it didn't work?

A. Yes singleplex show increase in fluorescence. but it didn't work when i mix three beads . My two singlex not working in multiplexing.

What bead kit from Luminex did you use to build your assay?

And the 2 single plex is not working in the multiplex due to high background so you don't see an increase in fluorescence? But the third single plex is working in the multiplex?

Yes exactly singlex is not working due to high background, there is no increase in fluorescence. Third one is working good . See the two graphs below having PCR product diltued from 10-100000. But its showing abrupt result in two singlex where as good decrease in third which I am saying is working.

I am using biorad beads for labelling cat no: MC10037-01 and protocol used given in luminex cook book at page no: 19.

Here's a silly question, what regions are these 3 beads on that your are using?

Another silly question, I'm assuming the probes sequences don't overlap?

While the one looks like it's working, it looks like you reach background at X3.

Did you check the multiplex with just the oligos? Instead of PCR product?

If everything was done correctly, I can't think why all 3 single plex will work while done separately, but 2 would completely fail when mixed together. (At most we should only see crossreactivity, if even that.) That high of a background makes me think that there are unspecific binding and/or excess biotin bound to the beads for some reason.

If you don't think it's any of the above, I would try a different sequence to bind to the beads for the 2 that are failing.

I know I ask a lot of repetative questions, but it's difficult to troubleshoot over the computer.

Q. what regions are these 3 beads on that your are using?

A. I am not using specific region just labellling three beads different type13,14 etc.

I'm assuming the probes sequences don't overlap?

Yes no overlapping

Q. While the one looks like it's working, it looks like you reach background at X3.

Did you check the multiplex with just the oligos? Instead of PCR product? NO check with PCR product.

Q.If everything was done correctly, I can't think why all 3 single plex will work while done separately, but 2 would completely fail when mixed together. (At most we should only see cross reactivity, if even that.) That high of a background makes me think that there are unspecific binding and/or excess biotin bound to the beads for some reason.

A. I want to correct unspecific binding and/or excess biotin but dont able to find it why it is occuring.

If you don't think it's any of the above, I would try a different sequence to bind to the beads for the 2 that are failing.

I cant use different sequence as I am taking already standardized multiplex primers which have been published.

Wait... if you are not using beads with specific regions, how can you multiplex? (The catalog number you provided is for beads in region 37.) For example, my company do this with antibodies (for protein detection), same idea. To multiplex 10 analytes, each antibody for a specific analyte is coated on a bead with a specific region. So in the multiplex, there are 10 different beads, each on a different region.

Yes you are right I am saying that for multiplex I am using region 13, 14 and 37.

You mentioned that these sequences were published, so the paper that you took these from did a multiplexing with the beads?

No not with beads. They do it with conventional PCR only showing multiple bands in agarose gel electrophoresis.

Another question, how big are the PCR products for the 3 assays? Are they all the about the same length for the one that worked versus the ones that didn't

No they are not of same size. one which are not working have size of 800 and 412 bp. The working pair has size of 451bp.

Um... those are pretty big for this type of assay. Usually, when we use anything with a probe (like the beads), the PCR product we aim for is fairly small `100 TO 200bp. Anyway... in case you haven't see the article below... it gives some good advice on designing multiplex Luminex arrays for PCR.

Article High Throughput Multiplex PCR and Probe-based Detection with...

Hi Kanisht Batra and Shen-An Hwang

Thank you for a stimulating conversation! Kanisht Batra , I’d recommend some in silico work to lay out primer and probe sequences and ordering formats, along with amplicon lengths. You’ll want to check that all of that preparation aligns with Luminex recommendations for optimum performance using multiplexed PCR and bead-based detection. Recommendations can be found in the updated xMAP Cookbook, accessible via this link: https://info.luminexcorp.com/en-us/research/download-the-xmap-cookbook

For additional support in troubleshooting, you can contact the Luminex support team for assistance at [email protected].

Thanks a lot Shen-An Hwang . I am changing the product size.

Heather Darby - Can you please tell what should be the PCR product size in multiplex format for efficient coupling in Luminex assay ?

Hi Kanisht Batra . I recommend amplicons