Single stranded molecules run crazy slow on a DNA Agilent Chip

[pmiguel]

Top panel, normal RNA nano ladder run on a single-strand assay Bioanalzyer chip (RNA nano). Sizes in nucleotides.

Bottom panel, same RNA nano molecular weight ladder run on a double-strand assay Bioanalyzer chip (High Sensitivity DNA). Sizes (incorrect) in base pairs.



The first 3 fragments run at approximately double their true molecular weight. That is 200, 500 and 1000 nucleotides run as 236, 428 and 902 base pairs. Then 2000 and 4000 nucleotide fragments run as 10 and 27 thousand bp. Well, that is my interpretation.

There could actually be some utility in this phenomenon for the lower MW molecules. If you have a mixture of ds and ss molecules and want to know their strand lengths, then you would get approximately the right answers. But if your are expecting them to run at their true MW then you might be confused by your results.

For the converse phenomenon on (double stranded DNA on an RNA chip runs faster than expected) please see my earlier thread:

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

[CC_seqanswers]

How about running ssDNA on a ds DNA chip? Is it gonna work fine?

[HESmith]

I noticed that the fluorescence units for your DNA chip graph are pretty high. We observe the same phenomenon you observe when the chip is overloaded, even with dsDNA. You might want to dilute the sample to the appropriate concentration (<10 ng/ul) and retest.

-Harold

[pmiguel]

How about running ssDNA on a ds DNA chip? Is it gonna work fine?

Same phenomenon, single stranded molecules migrate unexpectedly slowly with respect to double stranded molecules. See:

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

[pmiguel]

I noticed that the fluorescence units for your DNA chip graph are pretty high. We observe the same phenomenon you observe when the chip is overloaded, even with dsDNA. You might want to dilute the sample to the appropriate concentration (<10 ng/ul) and retest.

-Harold

The single stranded sample is just RNA (nano) ladder. So the concentration loaded is just 1 ng/ul. The fragment concentrations are in the same approximate range as the High Sensitivity Chip ladder fragment (~150 pg each). So I do not think the lane is overloaded.

Did you take a look at the converse result I saw when running dsDNA on an RNA chip?

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

[vasvale]

salt composition

may check with Agilent about effects of sample's salt/tween that change the migration

[pmiguel]

Hi vasvale,
Have you ever tried communicating with Agilent? Personally, I have not found it to be productive.

In any case, since we are loading the ladder from another chip type, it seems unlikely there is a difference in the salt/tween concentration between it and the double stranded ladder.

--
Phillip

[sehrrot]

Hi pmiguel.

I love this work! you are so...
I've never done this before but I used a dye of 1000DNA chip for HS chip and worked well.

[victorsor]

Hi pmiguel.

I love this work! you are so...
I've never done this before but I used a dye of 1000DNA chip for HS chip and worked well.

More over, if you run a 7500 chip with HS protocol and reagents, works as a HS chip.

[pmiguel]

More over, if you run a 7500 chip with HS protocol and reagents, works as a HS chip.

We used to do this quite a bit. But we started getting strange results -- like the instrument refused to run -- after a while. So we gave up on it.

--
Phillip

[pmiguel]

I've never done this before but I used a dye of 1000DNA chip for HS chip and worked well.

Could you go into this a bit more? Like why you did it and whether you still used the extra HS reagent that goes into its own well. Was the sensitivity still HS, even without using HS dye?

--
Phillip

[victorsor]

The single stranded sample is just RNA (nano) ladder. So the concentration loaded is just 1 ng/ul. The fragment concentrations are in the same approximate range as the High Sensitivity Chip ladder fragment (~150 pg each). So I do not think the lane is overloaded.

Did you take a look at the converse result I saw when running dsDNA on an RNA chip?

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

This is not true, concentration of NANO ladder is 150 ng/ul, about 30 ng for each peak. 1 ng/ul is the concentration of PICO ladder.

[victorsor]

Top panel, normal RNA nano ladder run on a single-strand assay Bioanalzyer chip (RNA nano). Sizes in nucleotides.

Bottom panel, same RNA nano molecular weight ladder run on a double-strand assay Bioanalyzer chip (High Sensitivity DNA). Sizes (incorrect) in base pairs.



The first 3 fragments run at approximately double their true molecular weight. That is 200, 500 and 1000 nucleotides run as 236, 428 and 902 base pairs. Then 2000 and 4000 nucleotide fragments run as 10 and 27 thousand bp. Well, that is my interpretation.

There could actually be some utility in this phenomenon for the lower MW molecules. If you have a mixture of ds and ss molecules and want to know their strand lengths, then you would get approximately the right answers. But if your are expecting them to run at their true MW then you might be confused by your results.

For the converse phenomenon on (double stranded DNA on an RNA chip runs faster than expected) please see my earlier thread:

http://seqanswers.com/forums/showthread.php?t=12501

--
Phillip

I'm not sure that the peak sizes are well assigned. Fluorescence intensity of high marker is aproximately the same that Low marker, so i think that is more probably that the "587 pb peak" is the High marker. Check at what time appears each peak.

[pmiguel]

I'm not sure that the peak sizes are well assigned. Fluorescence intensity of high marker is aproximately the same that Low marker, so i think that is more probably that the "587 pb peak" is the High marker. Check at what time appears each peak.

Hi victorsor,

The lowest peak is not part of the ladder -- it is spiked in separately and is a different size for the nano (25 nt) and the DNA HS (35 nt). If you look at the relative areas of all the other major peaks, they are reasonable.

Also looking at time, instead of MW would not necessarily help. The Agilent software actually alters the time to align the lanes. If you change the position of the upper or lower marker, you will find its reported "migration time" will change as well.

Thanks for your comments about the mass of the MW weight standards for the pico and nano chip. If true, then the chip was overloaded. I should run another one, but I am not sure when or if I will get around to it.

--
Phillip

[colicoli]

Hi Phillip,

There are a few differences between the different bioanalyzer kits, esp. the RNA vs. DNA. First, as you'd expect they use different dyes, and some dyes bind dsDNA better, others prefer ssDNA or RNA. Second, the RNA chip formulations can be somewhat denaturing; they will remove some but not all secondary structure. Often RNA or ssDNA runs more slowly on a DNA chip due to residual secondary structure, and dsDNA runs faster on an RNA chip because it is rod-like. Finally, the different protocols (esp. the HS protocols) may actually use different voltages and buffers. I think the targets can be "preconcentrated" by varying voltage in a low-ionic-strength buffer. This is one reason the sample should not have much salt in it.

Finally, two tips in case things are not running the way they used to: first, remove and scrub the pin electrode assembly under di-water, dry it and replace. The recommended rinse does not remove salt deposits as well as a good brushing under running water. Second, watch the expiry dates on the reagents, esp. the chips. You might expect that glass chips would last forever but there is plastic outgassing so it is not so simple.


Full disclosure: I actually work in R&D at Agilent, though not in groups related to sequencing or the bioanalyzer. But I've used the Bioanalyzer a lot, often for off-label functions. I've had good luck running mixtures of dsDNA and oligos on the small RNA chip, though I had to run parallel standards of known size and structure.