Difference between revisions of "Part:BBa K3113005"
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<h2>Characterization</h2>
 
<h2>Characterization</h2>
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<h3>Detected RNA levels correlates to the amount of cells monitored
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To show that our pipeline of RNA isolation and RT-qPCR works robustly and to determine the detection limit of our system, we performed a Log2 titration on cell density and measured exported mRNA in secreted VLPs and exosomes.
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        <img src="https://2019.igem.org/wiki/images/4/4b/T--Munich--Titration_Results.png" width="50%" class="figure-img img-fluid rounded" alt=" ">
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          <b>Figure 9: Correlation between FLuc mRNA isolated from vesicles dependent on number of transfected cells.</b>  The amount of transfected cells was titrated in a range of 25000 to 400 cells per 96-well and RNA was quantified via RT-qPCR. Quantification was done via standard curve measurements and assuming a confluent well with 350.000 HEK293T cells/cm2 at the time of harvesting. n = 2.
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The amount of detected Fluc mRNA cargo in exosomes or VLPs decreases linearly with decreasing cell number (figure 9). Robust detection of our target RNA is possible down to 400 or 800 cells for exosomes and VLPs, respectively, before no clear differentiation to the next-smaller cell number is possible anymore. This proves that our system is highly sensitive, and our RNA extraction methods including RT-qPCR work properly and stably.
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<figure class="figure">
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        <img src="https://2019.igem.org/wiki/images/e/e4/T--Munich--qPCR_Sequencing.png" width="50%" class="figure-img img-fluid rounded" alt=" ">
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        <figcaption style="font-size: 80%">
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            <b>Figure 10: </b>Verification of secreted Fluc mRNA by sequencing. Fluc mRNA was purifed with chloroform/phenol isolation, reverse trancribed to cDNA and amplified by qPCR. The qPCR product was further isolated from an agarose gel for sanger sequencing. The sequencing results were successfully aligned to its reference in genious.
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Even though we were confident that high-quality transcripts could be exported, we assessed whether we can sequence our cargo after running the full RNA purification and quantification pipeline. We purified the qPCR product on an agarose gel and sent it for Sanger sequencing. The sequencing result clearly proves that the amplified FLuc-cDNA is intact and pure enough for sequencing (Figure 10), demonstrating the integrity of our exported cargo. Furthermore, no mutation can be found indicating that RT and qPCR polymerase work properly. This opens the possibility to detect multiple transcripts exported in exosomes or VLPs. Since RNA is made up of 4 bases, the encoding potential of RNA is therefore given by 4n for an RNA strand with n nucleotides. Even with short RNA molecules exported, the information potentially delivered by ALiVE greatly surpasses any current non-invasive gene reporter assays.
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Revision as of 00:27, 22 October 2019


FLuc

This DNA sequence codes for the firefly luciferase. The firefly luciferase (FLuc) is a light-emitting enzyme. This sequence has its origin in the organism Photinus pyralis.

Usage

The readout of ALiVE is based on RNA. We used FLuc as message loaded into the vesicles. This had the advantage, that we could test our vesicles for passive transfection. The cells treated with our vesicle could be examined for passive transfection via a FLuc assay.

Biology

FLuc is an oxidative enzyme, producing bioluminescence in the presence of the substrate luciferin and oxygen.

Characterization

Detected RNA levels correlates to the amount of cells monitored

To show that our pipeline of RNA isolation and RT-qPCR works robustly and to determine the detection limit of our system, we performed a Log2 titration on cell density and measured exported mRNA in secreted VLPs and exosomes.

Figure 9: Correlation between FLuc mRNA isolated from vesicles dependent on number of transfected cells. The amount of transfected cells was titrated in a range of 25000 to 400 cells per 96-well and RNA was quantified via RT-qPCR. Quantification was done via standard curve measurements and assuming a confluent well with 350.000 HEK293T cells/cm2 at the time of harvesting. n = 2.

The amount of detected Fluc mRNA cargo in exosomes or VLPs decreases linearly with decreasing cell number (figure 9). Robust detection of our target RNA is possible down to 400 or 800 cells for exosomes and VLPs, respectively, before no clear differentiation to the next-smaller cell number is possible anymore. This proves that our system is highly sensitive, and our RNA extraction methods including RT-qPCR work properly and stably.

Figure 10: Verification of secreted Fluc mRNA by sequencing. Fluc mRNA was purifed with chloroform/phenol isolation, reverse trancribed to cDNA and amplified by qPCR. The qPCR product was further isolated from an agarose gel for sanger sequencing. The sequencing results were successfully aligned to its reference in genious.

Even though we were confident that high-quality transcripts could be exported, we assessed whether we can sequence our cargo after running the full RNA purification and quantification pipeline. We purified the qPCR product on an agarose gel and sent it for Sanger sequencing. The sequencing result clearly proves that the amplified FLuc-cDNA is intact and pure enough for sequencing (Figure 10), demonstrating the integrity of our exported cargo. Furthermore, no mutation can be found indicating that RT and qPCR polymerase work properly. This opens the possibility to detect multiple transcripts exported in exosomes or VLPs. Since RNA is made up of 4 bases, the encoding potential of RNA is therefore given by 4n for an RNA strand with n nucleotides. Even with short RNA molecules exported, the information potentially delivered by ALiVE greatly surpasses any current non-invasive gene reporter assays.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage