Bioanalyzer or Tapestation Traces

[Justin Wong]

I was interested in potentially seeing Bioanalyzer or Tapestation traces from other folks if possible.  In particular, if anyone has any insight onto what I can expect from laser-capture microscopy (LCM) on FFPE samples, that would be great.  We are looking to understand some of the variability we see between samples, and to see if what we are seeing is normal.

[Olivier Harismendy]

we do not QC the RNA since we go straight from cap to cDNA. I think that is the strength of the Smart-3seq protocol. 

On Feb 21, 2019, at 11:13, Justin Wong <justin....@gmail.com> wrote:

I was interested in potentially seeing Bioanalyzer or Tapestation traces from other folks if possible.  In particular, if anyone has any insight onto what I can expect from laser-capture microscopy (LCM) on FFPE samples, that would be great.  We are looking to understand some of the variability we see between samples, and to see if what we are seeing is normal.

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[Justin Wong]

Yes, sorry, I was referring to the traces of the final libraries.  

[chunfa...@gmail.com]

This is the tapestation pictures for LCM samples from FFPE.  E1 is the primer dimers showing up.

[Olivier Harismendy]

Are those traces from pooled or single libraries ?

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<tapestation for FFPE LCM.png>

[Justin Wong]

We are presently generating traces from single libraries - I've attached ours as a reference for what we have been seeing.  We have sometimes seen that the primer-dimer doesn't get removed that well (see attached PNG) - we have been using 0.7X for SPRI selection (once, for single libraries).  And I know that the original protocol calls for 0.6X.  Does it make at all sense to move to somewhere inbetween which based on the chart seems to better target a 200-bp cutoff?

We see what could be overamplification of the primer-dimer, but I'm wary that driving the # of PCR cycles down too far will result in no sequencable library.

~Justin

[Justin Wong]

Do any of you who have run sequencing on these, think these could be sequenced?

[Chunfang zhu]

The 0.7X SPRI beads selection is enough for most of the library preparation. For the library with higher primer dimers, you can check the ratio of 165-500bp / <50-165bp. If the ration > 3, the qPCR of the library is more than 1nM. It still can be sequenced in my experience.

 

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[jm googlecalendar]

I am new to the party and am jazzed by this method, though in working through the steps I am unsure, exactly, about RNA quantification.  Dr. Ohari (above) does not QC RNA in the cap, yet in the protocol (Experimental design considerations, page 1) it says:  "an accurate measurement of that amount is crucial; fluorometry...is strongly recommended."  Moreover, in the next section (Choosing the number of PCR cycles) is specifies "total RNA per sample," which implies that RNA must be estimated for an accurate calculation.  If RNA is quantified by fluorometry, it seems the time to do that is in the lysate supernatant before the RT step, which I would like to confirm.  I have also read that it is important to know the characteristics of the amplified library, which suggests to me Kapa PCR with a Bioanalyzer chaser (for size distribution).  Knowing exactly when to assay and exactly what to look for (diluting concentrated samples, for example) would be of great value.  Thanks for those who have done the hard work on this approach.  j

[Joe Foley]

There are two versions of the protocol: either you start from RNA that you've isolated somehow and quantified, or you start from tissue on an LCM cap. If you have RNA, you should quantify the stocks with fluorometry to get reproducible results with the right number of PCR cycles. However, the protocol for LCM tissue directly consumes the entire tissue sample so there isn't any quantification to do; you just have to calibrate your PCR cycles for roughly the same amount of the same kind of tissue, then expect more variation in the yields and do plenty of replicates to protect against occasional failures.

For library quantification I recommend TaqMan qPCR (https://openwetware.org/wiki/Quantifying_sequence_libraries_with_qPCR) instead of SYBR since the length distributions can be a little messy and I don't have confidence in electrophoresis (Bioanalyzer/TapeStation/Fragment analyzer) to estimate the average molecule length, which is required to normalize SYBR data, but electrophoresis is still required for overall QC.

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[jm googlecalendar]

Thanks for your quick response.

We will begin comparing fresh tissue-derived RNA with comparable fixed-embedded-sectioned-LCM material (bulk) and measure how much RNA there is as a starter.  Based on that, as you suggest, we can calibrate our PCR cycles.  Thanks.

We are, however, using a core facility with the Zeiss PALM system rather than Acturus.  Might you have any experience or information on that platform?

[Joe Foley]

We will begin comparing fresh tissue-derived RNA with comparable fixed-embedded-sectioned-LCM material (bulk) and measure how much RNA there is as a starter.  Based on that, as you suggest, we can calibrate our PCR cycles. 

If possible I would recommend calibrating the protocol on non-precious tissue samples the same way you would use it on the precious ones. That also gives you the opportunity for a test run to make sure everything is working. If you add an extra RNA isolation step and use the non-LCM protocol for only the calibration samples, you're comparing the unknown efficiency of your RNA isolation protocol with the unknown (but probably lower and more variable) yield of the lysis in the Smart-3SEQ LCM protocol and mixing that with any difference in efficiency between the LCM and non-LCM protocols.

We are, however, using a core facility with the Zeiss PALM system rather than Acturus.  Might you have any experience or information on that platform?

I don't but our LCM protocol is specific for the capture "caps" in the Arcturus and Fluidigm LCM systems. You may need to adapt it for the physical design of the analogous parts in your system, though hopefully the same reaction mixes and thermal-cycler programs will still work.

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[jm googlecalendar]

Your point about Taqman quantification is well taken.  I am wondering if you have any thoughts about using the integrated metric DV200 (% of fragments >200 nt) quoted by Illumina that reportedly is a better predictor of library yield compared to RIN.

https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf

j

[Joe Foley]

I haven't looked much at different RNA qualities, since the protocol works even on very degraded RNA. My intuition is that random RNA fragmentation slows down asymptotically as fragments get short, since longer molecules have more places to break, so from FFPE we've probably already been looking at the lowest RNA integrity you're likely to deal with.

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