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I am currently doing the ground work for a SELEX experiment and I am attempting to verify my standards for the starting library. I ran a reaction with samples containing 50, 5, 0.5, and 0.05ng of my starting library and the samples for the 5, 0.5, and 0.05 appear to amplify as expected, but whenever I run a sample containing 50ng of my starter library, the curves start around -2000 RFU and peak at 0 RFU. Has anyone seen this behavior before?

EDIT: Added annotated graph Method: SYBR Green System: Bio-rad CFX384 Software: Bro-rad CFX Manager

enter image description here

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    $\begingroup$ Can you please label your curves to make it clear which is which? Also, how are you computing RFU? Getting negative fluorescence usually indicates a process failure of some sort. $\endgroup$
    – jakebeal
    Jun 15 at 19:45
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    $\begingroup$ @jakebeal My apologies, I should have annotated it from the start. I've updated the post to include the annotated graph and more information. I am reading RFU with SYBR Green on a Bio-Rad CFX384. I've performed this assay several times with fresh dilutions of library DNA and the results seem to be the same each time. $\endgroup$ Jun 15 at 23:52
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    $\begingroup$ I'm not too familiar with SELEX, but just looking at the curves, it seems like a baseline normalization issue. The Bio-Rad software that I use automatically normalizes amplification curves to a baseline average from the first few cycles. Since your 50ng wells were hot from the start, it looks like it took the saturated endpoint fluorescence as baseline, and normalized the rest of the curve to that. You can turn normalization off in the software, but probably still won't be able to get a good Cq value for the 50ng curves. $\endgroup$
    – MikeyC
    Jun 16 at 13:41
  • $\begingroup$ @MikeyC This does seem like the most likely explanation. The only thing that might make a difference in this regard is that I did run a no-template control which was stable at 0 throughout the run. It is not pictured here because it covered up a portion of the 50ng sample curve. I am new to this system so I am not totally sure how it goes about calculating a baseline. Thank you for your input. $\endgroup$ Jun 16 at 20:30
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    $\begingroup$ @jakebeal The no-template control doesn't factor in. The software determines a different baseline for each well, which it subtracts from that well's fluorescence readings throughout the run so they can all lineup at 0 for the first few cycles. It's just not getting a usable baseline from those wells, so it's subtracting the saturated fluorescence instead. You can disable baseline subtraction under the Settings menu in your CFX manager software. Only problem is, you can't set a threshold for Cq determination without baseline subtraction. $\endgroup$
    – MikeyC
    Jun 16 at 22:23

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