Team:EPFL/Notebook-Detection

iGEM EPFL 2018

Notebook - Follow-up: miRNA

This page collects the notebooks of the Follow-up part of our project specifically dedicated to miRNA. The experiments are sorted according to the three main subparts of this section, and then by week (inside each subpart).

Probe preparation and Rolling Circle Amplification

This part comprises all the steps from the phosporylation of the linearized probes, to the ligation and the Rolling Circle Amplification (RCA). More information is available on the protocols of probe preparation and RCA.




Week 3 (23/07/18 - 29/07/18)


THURSDAY, 26/07/2018

Phosphorylation and ligation of Probe 2

Aim:

The aim is to prepare the probes by phosphorylating and ligating of the oligos we received from IDT. We first start to test probe 2, that we know from literature is correctly amplified, to verify that the protocol is working. Then, we plan to repeat both steps also for the other probes.

Description:

The oligos we ordered arrived dried, so we needed to resuspend them to 100 μM by using DEPC-treated water.

We first decided to make the phosphorylation and the ligation with probe 2, because among the ones proposed by Qiu et al., it was proved to be the most efficient one with respect to amplification.

The probes were ordered from IDT as non-phosphorylated oligos, because they were significantly less expensive than 5'-phosphorylated oligos with the same sequence. As a consequence, in order to achieve the ligation, we will always have to first phosphorylate our oligonucleotides with T4 Polynucleotide Kinase (PNK).

The sequence of the probe 2 is: 5'-ACCTCATTGTATAGCCCCCCCCTGAGGTAGTAGGTTGCCCAACTATACAACCTACT-3'.

During this step, we used (and we will have to use for every phosphorylation) T4 ligase buffer - that contains ATP - instead of PNK buffer, because the nucleotide is required to transfer a phosphate group to the 5' end of the oligo. We point out that the phosphorylation step cannot be verified e.g. by means of an agarose gel because of course the phosphorylated and non-phosphorylated oligos actually have the same length.

The ligation was done with T4 DNA ligase, and all the non-ligated probes were digested by using Exonuclease I and Exonuclease III, two 3'->5' exonucleases. The complete protocol for the preparation of the probes is shown here.

We decided to put the exonucleases without any buffer, as suggested in the protocol from Qiu et al.. Nonetheless, we were not sure whether the activity of the proteins was sufficient to cleave all the non-ligated oligos. Therefore, we decided to assess our sample on a 1.5% agarose gel (100 V, 30 min) together with a negative control, as described in the table below:

Sample T4 ligase Exonucleases
Ligated probe 2 + +
Negative control - -

If a band at 56bp (size of the probe) is also shown in the lane for the negative control it means that the Exonucleases did not work fine and that the protocol needs to be modified.

The agarose gel also provides information on the phosphorylation: indeed, if no any band is shown for our sample either, it means the oligo was not ligated and that the phosphorylation did not succeed.

Results:
Image
Agarose gel for the ligation of the probe 2.

One band was shown for the sample where we add both ligase and exonucleases. We cannot tell the exact size of this band because the shortest band of our ladder was 100bp long. Nonetheless, from this result we can be confident that the band is indeed the ligated probe 2.

Moreover, for the negative control, no band was shown in the gel, meaning that all the non-ligated oligos were actually digested by the enzymes.

Discussion

We can see that 1μl of Exonuclease I and III cleave all the non-ligated probes (100μM) in the second sample. As a consequence, we can conclude that the amount of nucleases we put is sufficient.

Also, the results confirm that the phosphorylation and the ligation worked fine.


SATURDAY, 28/07/18

Rolling Circle Amplification of probe 2 - Trial 1

Aim:

The aim is to amplify the probe 2 by using different concentration of "miRNA" : 10μM, 1μM, 100nM, 10nM. The amplification is then checked by using an agarose gel.

Description:

As of today, we have not received the RNA oligonucleotide for let-7a yet. Nonetheless, we also ordered a DNA oligo with same sequence as let-7a (but with "thymine" instead of "uracil"): for the first part of our project, we will work with this DNA oligo instead of the actual RNA sequence. The idea is to check whether the amplification also works with DNA, which could be highly convenient as a proof of concept because of the high prices of RNA oligonucleotides (10-fold more expensive than a DNA oligonucleotide with the same sequence).

Moreover, our idea is also validated from literature, since Rauzan et al. have shown the thermodynamics of the DNA/DNA duplex formation is similar to RNA/DNA duplex.

The DNA "miRNA" oligonucleotide (later also referred to as "fake miRNA") was first of all resuspended to 100 μM by adding Ultra-pure water. We then performed serial dilutions from the DNA solution stock to 10 μM, 1μM, 100nM and 10nM.

After, we performed Rolling Circle Amplification of Probe 2 as from the protocol, with 10 nM fake miRNA. The reaction product was then tested by means of a 0.7% agarose gel (100V, 40 min)

Results:
Image
Rolling Circle Amplification of probe 2, with 1μM of the DNA oligo for let-7a.
Discussion:

The results are not so clear but it appears that there is a band in the well, due to the size of the amplicons. We will perform the same experiment again so as to be sure of the results.


SUNDAY, 29/07/18

Rolling Circle Amplification of probe 2 - Trial 2

Description:

We performed the Rolling Circle Amplification again for different concentrations of fake miRNA (DNA) solution, namely 10nM, 100nM, 1μM and 10μM.

Results:
Image
Rolling Circle Amplification of probe 2, with different concentrations of the DNA oligo for let-7a.

The results might be not straightforward to interpret. Indeed, as a product of the RCA reaction we expect to have a very large amplicon (roughly 70 kb long): because of its big size, such amplicon can therefore migrate very slowly in the gel and remain very close to the wells. This is indeed what we mostly can observe here, with very thin bands near the wells. In addition, we also notice smears for the samples with higher concentration of miRNA.

Discussion:

Again, due to the high size of the amplicon, agarose gels might not be the most suitable assay to definitively verify that the amplification worked fine for the different concentration of probes, even if our gel seems to show consistent bands very close to the wells, as expected.

Moreover, our gel is sufficiently comparable to gels shown by other groups working on Rolling Circle Amplification, as the one presented below:

Image
Agarose gel analysis of RCA product, indicated by the arrow. Reproduced from Takahashi et al. (Figure 4C).




Week 4 (30/07/18 - 05/08/18)


MONDAY, 30/07/18

Preparation of probes from 3 to 10

Aim:

The aim is to prepare the other probes, from 3 to 10, now that we know that the phosphorylation and the ligation work. The preparation of probe 1 will be done in the next days.

Description:

After following the protocols for ligation and phosphorylation, we tested the different probes by means of a 1.5% agarose gel (100V, 30 min). The negative control was not done here.

Results:
Image
Agarose gel after ligation for probes from 3 to 10

As expected we can observe that there is a band for all the probes. For probe 3, the band has a very low intensity. Also, the band for probes 6 and 9 seems to be more smeared and to extend up to a higher size (>100bp).

Discussion:

In general, we were able to show the all the probes from 3 to 10 could successfully be ligated. The low intensity of probe 3 means that the ligation yielded a low amount of properly-ligated probes. For what concerns instead probes 6 and 9, both of them have a G-rich loop which would explain the unexpected size: the smear might indeed be due to the formation of duplexes of misligated probes. We will therefore not use probes 6 and 9 for the Cas12a assay.


TUESDAY, 31/07/18

Rolling Circle Amplification of probes 3 and 4

Today we proceeded to amplify the probes 3 and 4 and to test whether the RCA reaction could work well enough with different concentrations of miRNA (DNA) : 1μM, 100nM, 10nM, 1nM. We ran a 0.7% agarose gel (at 120 V, 45 min), but unfortunately we broke the gel prior to put it in the UV transilluminator. As a consequence, we need to make the electrophoresis again tomorrow, to be sure that the amplification actually worked. By putting a sufficient amount of RCA product in the wells of the gel we actually expect to see a smear for a very wide range of sizes, because the size of the amplicon should depend on the time when the duplex phi29-probe is formed (if we do not assume that all the duplexes are formed at the same time).


WEDNESDAY, 01/08/18

Rolling Circle Amplification of probes 3 and 4

Aim:

The aim is to perform the RCA of the other probes, in order to be sure that the results we got for probe 2 can be replicated also with other probes. We first amplified probes 3 and 4.

Description:

As usual, we followed the protocol for RCA, then verified the products by means of a 0.5% agarose gel (100V, 1h). We tested two different ladders (2-log purple ladder - highest band at 1000kb, on the right in the image -, and 100bp ladder - left in the image), because the first ladder was expired and so we were not sure if it could be used.

Results:
Image
Rolling Circle amplification of the probe 3 and 4.

It seems again that there are some bands very close to the well for both of the probes and for the different concentration of let-7a (DNA).

Discussion:

We can now be rather confident the RCA works for these probes. A more accurate way to analyze the RCA products would be to use the intercalating dye SYBR Green I; nonetheless, this reagent is very expensive and therefore we do not plan to buy it for the moment.


THURSDAY, 02/08/18

Rolling Circle Amplification of probes 5 to 10

Aim:

The aim is to make the amplification of the other probes, this time probes from 5 to 10.

Description:

The assessment of the experiment has also been done with a 0.5% gel. As our electrophoresis device is small we could only use the small comb that produces only ten wells. As a consequence, we had to reduce the number of different let-7a DNA concentrations and we could not represent the results for all the probes in one gel.

Results:
Image Image
Agarose gel for the RCA products for different concentrations of let-7a DNA, with probes 5-6-7-8 (on the left) and probes 9 and 10 (on the right).
Discussion:

Also in this case the results suggest that the amplification for probes 5 to 9 was successful. Surprisingly, a band was also shown for probe 10, which was not expected, since probe 10 was designed to incorporate a mismatch with respect to the miRNA sequence; this implies that a non-complete match between the probe and the miRNA is sufficient to trigger amplification. This might be a positive finding (implying the need of less stringent constraints on the probe design), but also needs carefully considerations: the probes need indeed to be sensitive enough to bind only to the target miRNA and not also to other miRNAs with similar sequences.




Week 6 (13/08/18 - 19/08/18)


TUESDAY, 14/08/18

Rolling Circle Amplification of probe 1

Aim:

We want to check if the amplification works also with probe 1, which is, among the probes we designed ourselves, the one which we believe might lead to the best result.

Description:

We did the phosphorylation and the ligation of probe 1 but we decided not to make another gel because we already validated our protocol multiple times.

It has to be noted that it is the last time we will use let-7a DNA. Indeed, we finally received our RNA oligonucleotide and we also want to test if our RCA protocol works also with actual RNA.

The gel analysis was performed with a 0.5 % agarose at 120V for 75min.

Results:
Image
Agarose gel for the RCA product of probe 1
Discussion:

The image is very unclear, probably also because of a mistake in the UV exposure of the gel. Furthermore, we wanted to run the gel for a longer time to see whether the amplicons could travel in the gel further from the wells. This nonetheless almost resulted in the removal of the ladder from the gel (we can only see the larger bands), but did not have a positive effect on the amplicons.





Week 8 (27/08/18 - 02/09/18)


TUESDAY, 28/08/18

Rolling Circle Amplification using miRNA :

Aim:

The goal is to make the amplification using the actual RNA let-7a oligonucleotide instead of its DNA copy.

Description:

Now that we received let-7a under RNA form, we can start to work with it and to make sure that the amplification also works with actual RNA. We decided to assess the amplification using probe 1, as it is the probe that we currently use for Cas12a assay.

The gel is as usual a 0.5% agarose gel at 100V for 60min.


As we use RNA now, we have to be careful and to work in a RNase Free Zone.

Results:
Image
Agarose gel of the RCA products of probe 1 with different concentrations of miRNA

We were able to see bands in the wells, but as usual the results were not very clear: in particular, we were able to see faded bands close to the well for all the four concentrations, even if they were brighter for the two highest concentrations.

Discussion:

Even if the results are not very accurate, we can, by comparing this gel with the ones from our previous experiments, qualitatively infer that the amplification works with miRNA as well. Once more, this confirms to us that agarose gels are not the most suitable assay to test whether RCA works, and that we might indeed need to buy SYBR Green I to have more quantitative information on the RCA products.


WEDNESDAY, 29/08/18

Today we first of all proceeded to aliquote the different dilutions of the let-7a: the 100 μL tubes with each of the four meaningful concentrations (1 μM, 100 nM, 10 nM and 1 nM) were divided into 3 smaller tubes of 20 μL, with the remaining 40 μL in the original tubes. All the tubes were stored back at -65°C, except for 1 small 1 μM one which was partly directly used for experiment of today.


Rolling Circle Amplification with different incubation times.

Aim:

The aim was to have further confirmation of the amplification being working, in addition to the gels from the previous experiments. We want to prepare different samples that have the same probe and the same amount of RNA, but we will vary the incubation time (i.e. the step of the protocol at 37°C, before the denaturation step at 65°C), from 5 min to 2h.

Description:

In this experiment we wanted to repeat the amplification with probe 1, but with different incubation times, in order to check on the gel whether shifted bands appeared (to have a further proof of the RCA being working). We therefore tested RCA for the 1 μM let-7a in 4 different tubes, incubating those latter respectively for 2 hours, 1 hour, 25 minutes and 5 minutes (just pausing and resuming the ThermoCycler to insert the samples), with of course also the final 10 min 65°C denaturation step. We then ran a gel of the obtained amplicons with the standard parameters (0.7%, 120 V, 40 minutes).

Results:
Image
Agarose gel of RCA products (probe 1, 1 μM let-7a) with different incubation times

We can see smears for all the lanes, from roughly 50bp (not precise because our ladder is adapted to high sizes of DNA) to a very high size (that we cannot determine with our ladder) for 1h and 2h incubation times.

We can also see a large band at 25 min (the size cannot be determined accurately because of the exponential behaviour of the ladder) and bright bands in the wells for 1h and 2h, as well as other bands of various sizes.

Discussion:

First of all, we can say that the amplification is initiated pretty quickly because we can see a smear already at 5 min. Also, in this lane, there is a pretty bright band at 2kb. The rate of the phi29 polymerase is 2280 nt/min at 30°C (the rate at 37°C is not provided by NEB but we can assume that it is around 2000 nt/min): therefore, we can conclude that the binding of miRNA to the probe and the binding of the polymerase to the miRNA-probe complex occur within very little time from each other.

These results give further proof that the amplification actually worked properly.


Conclusion for this part

For the moment, the different assays that we performed allow us to qualitatively infer that our protocol for Rolling Circle Amplification actually works with our probes. Nonetheless, real-time measurements of SYBR Green I fluorescence activity might be needed in order to quantitatively verify the RCA products.