From the Lab

A second glance at disease response in tomato plants

Hello and welcome back to an update on my lab work! If you have come here without reading my first post you can find it here.

Since the last post

The last time I wrote about my lab work we had just finished RNA extraction and reverse transcription of the validation samples and were going to move on to the same process with our own samples. We have completed this and have also run all qPCR for the validation and our own samples. That involved a lot of pipetting but it was made easier through the use of an electronic pipette which takes up more than you need per well and ejects a set amount each time you click it.

csm_72.1985.202_3902_2e2ccc523a
Figure 1 384 well Lightcycler Plate[1]
In figure 1 you can see one of the plates that we had to pipette into for our qPCR. It took us over an hour to pipette each plate with the master mix (a mix of the SYBR green which fluoresces and is used to record the expression of the gene in question, the primer and water) and each sample (figure 2a and 2b).

IMG_2641IMG_2640

Figure 2a Plate design for validation samples. Figure 2b Plate design for our samples. The top left show the pool; a collection of samples at different dilution levels which are used to compare to the other results. NTC at the bottom right stands for non-template control which is the master mix plus water instead of cDNA to check that the chemicals weren’t contaminated.

 

Data analysis

Once pipetted the plate is run in the lightcycler for 1 hour and produces analysable data of when different samples fluoresced and what their melting temperature was, which is an indicator that a single product was being detected (figure 3a and 3b). Any samples that don’t have a single product will be removed from our analysis.

jarjar1

Figure 3a Melt curves for primer Jar1 showing two detected products and 3b where only one product is detected. Fluorescence is shown on the y-axis[3].

The Lightcycler also gives a Cq value which is the cycle where fluorescence exceeds that of the background fluorescence[4]. We are going to use this data to compare it to a reference gene, ours is actin, which has stable expression in all cells at all times. This comparison will tell us whether the gene has been up- or down-regulated in that sample.

What are we working on now?

While we are analysing our data for gene expression, we are also working hard to repeat all of the processes mentioned in this and in my first post on our second set of plants. 

IMG_2634
Figure 4 
Supporting our plants by attaching them to the stakes[5]

Last night (Friday) we re-staked and removed side shoots from our second set of plants which had grown a lot since we first did it (figure 4). We then zapped them with their first dose of UV-C. They will be zapped again on Sunday and Tuesday too. White light can undo the effect of the UV-C treatment so we have to wait until it gets dark before we can start our work.

It is going to be a busy few weeks to get everything done before our lab time ends on 7th April!

References

[1] SARSTEDT equipment providers https://www.sarstedt.com/en/products/laboratory/pcr-molecular-biology/pcr-plates/product/72.1985.202/

[2] Own image, plate design by Hannah Blyth.

[3] Own image using LightCycler 480 software7

[4] https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/06/15/qpcr-terminology-what-does-it-mean-

[5] Own image

Advertisements

Leave a Reply

Please log in using one of these methods to post your comment:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s