MED5484 Advanced Research Skills

Laboratory classes

Summative Assessments

Portfolio 25%

The student will be asked to provide a reflective portfolio notebook (e.g., lab book) detailing a summary of the methods employed in the laboratory sessions, problems encountered including appropriate solutions and key learning points.

Report 50%

The students will be required to provide a written report (ca. 2500 words), detailing methodologies used, implementation and analysis of data.

Oral Presentation 25%

The student will present a technical report of this exercise to their peers and supervisory staff. This will be in the form. of a short “flash-talk” which will be no longer than 5 minutes length. This format will be similar to that of a “Three-Minute-Thesis” talk which is commonly used at postgraduate level (e.g., PhD) in Universities.

Course overview

The aim of the MED5484 Advanced Research Skills module is to build on your understanding of basic laboratory skills gained in semester 1, allowing you to learn more in-depth techniques that can readily be utilised for microbiological and immunological research in oral sciences.

Week 1 – Tuesday 28^th January 2025

The first week will involve an introductory session led by Dr Brown, that will introduce you to the module, highlighting the purpose of the laboratory sessions, the methods to be used and an intro to the summative assessments.

Week 2 – Tuesday 4^th February 2025

All steps in red are for you to think about in relation to your lab book, lab report and/or presentation. In addition, all dilution steps are for YOU to work out. Any issues, ask for help from the teaching staff. Check all final calculations with a member of teaching staff.

DNA extraction

DNA will be extracted from PBS samples that mimic saliva. These samples will contain a range of microorganisms associated with oral health and a range of different oral diseases. These diseases will include oral candidiasis and periodontitis, whilst some samples will be representative of individuals with good oral health. There will be a total of 72 samples for the class to extract from. The “composition” of each sample will give an indication as to what disease the patients may or may not have.  Students will work in pairs and everyone will be required to extract DNA from their own samples (4 samples per person), and this will be used for qPCR (week 3). DNA will be extracted using the QIAamp DNA Mini Kit, according to manufacturer’s instructions (detailed below).

The DNA extraction protocol contains several different buffers and enzymes. Each of which has an important role in allowing sufficient extraction of DNA from the microbial cells. The kit we use for DNA extraction is purchased from Qiagen and contains the following:  

1) AL buffer

2) ATL buffer containing Proteinase K

3) AW1/2 buffers

4) AE buffer

Think about why these buffers are used, what are their functions in aiding the process of DNA extraction?

o Firstly, microbial cells will be pelleted by centrifugation at full speed for 10 minutes. Once pelleted, the supernatant will be carefully removed and discarded. This will be demonstrated by the supervisors if necessary – it is essential not to remove or dislodge any of the pellet.

o Next, prepare a working solution of proteinase K in ATL buffer. The proteinase K solution provided is 100% concentrated. The working solution concentration is 10%. You need a final volume of 900 uL.

o Add 200uL of the ATL/Proteinase K to your pellets and incubate at 56°C for 10 minutes. ATL Buffer may crystallise whilst in storage, therefore, needs to be heated up to decrystallize before use.

o Next, add 200μL AL buffer and incubate at 70°C for 10 min. Following incubation, add 200μL of 100% ethanol and transfer to spin column. What is the purpose of the ethanol and of the spin column? For this step, be careful when transferring the mixture. This will be demonstrated by the supervisors if necessary, as the solution may be foamy from the addition of the ethanol.

o Once transferred, centrifuge at 6000 x g for 1 minute then place column in new collection tube and add 500μL of AW1 buffer.

o Centrifuge again at 6000 x g for 1 minute then replace the collection tube again. Add 500μL of AW2 buffer and centrifuge at 12,000 x g for 3 minutes. Replace the collection tube again and centrifuge at 12,000 x g for 1 more minute. Why do you think we do this?

o Place column in 1.5mL RNAse-free Eppendorf and add 100μL AE buffer directly onto membrane. Incubate at room temperature for 5 minutes. Centrifuge 6000 x g for 1 minute.

o If available to use, and dependent on time constraints, your DNA concentrations can now be quantified using a Nanodrop – it is important to note concentrations and quality of your DNA samples in your lab book. Why do you think we do this? Why is the DNA yield of relevance here?

o Store the eluted DNA in the freezer until week 3 (qPCR). The teaching staff will provide a box for this and we will store samples at the dental school until next week.      

Week 3 – Tuesday 11^th February 2025

Quantitative PCR

For week 3 you will be expected to create and run a qPCR plate using the DNA extracted from the previous week. This will allow you to determine the colony forming equivalents for a range of different microorganisms in the “saliva” samples, allowing you to “diagnose” if the patient will likely have a disease or not depending on the composition of the microbiota from the sample. For this we will use a technique called fluorescent dye-based real time quantitative PCR (Figure 1), something you have encountered in the scientific publications in your journal club sessions. In short, the qPCR process will amplify the fluorescently “tagged” DNA in your sample using primers specific for the different microorganisms, resulting in levels that can be detected above the threshold of the machine.

Figure 1 – basic principles of fluorescent based qPCR

1) Firstly, a mastermix needs to be prepared using the SYBR green mastermix (the dye that is utilised for quantifying the DNA levels, including nucleotide bases to generate new DNA strands), forward and reverse primers for your organism of interest (to amplify specific regions of DNA for that given organism) and nucleotide free water. This will be combined with the DNA you extracted from the previous weeks “saliva” samples to run in the qPCR machine.

2) Each group will be asked to create a series of mastermixes for a range of different microorganisms (Streptococcus spp, F. nucleatum, P. gingivalis and C. albicans). This will be clarified on the day, and it will give everyone the opportunity to make mastermixes. In addition to running their own samples on the qPCR plates, each group will be given several “standards” or “positive controls”. Why do you think this is necessary?

3) It is the responsibility of each student in their group to prepare two microorganisms mastermixes. You will have a total of four per group, one for each microorganism. To do this prepare ONE mastermix to a final volume of 600 uL. SYBR green is 2X concentrated, and needs to be diluted to a 1X working solution. Primer sets are at a concentration of 100 µM. The working concentration of primers should be 5 µM. The remaining volume should include nucleotide free water. Repeat this step with the other THREE mastermixes, ensuring the right primers are used for each.      

*S1, S2, S3, S4 refers to sample 1, 2, 3 and 4 (ALL SAMPLES MUST BE RAN IN DUPLICATE)

Microorganism 1 (Candida albicans)

Microorganism 2 (Streptococcus spp.)

Microorganism 3 (Fusobacterium nucleatum)

Microorganism 4 (Porphyromonas gingivalis)

5) Once the mastermixes have been prepared, each student is required to add 19 µL of the mastermix for their designated microorganism to each appropriate well of a MicroAmp fast-optical 96-well, 0.1 ml reaction plate – follow the plate plan above as a template. For example, if you have prepared the mastermix for C. albicans, you will add 19 µL of this to every well in rows A and B e.g., this should be added to all wells, including the positive and negative controls in that row. The same process is repeated for the other three microorganisms.

6) Once the mastermixes are all added, you can start adding the samples. Remember these will be added in duplicate for all your samples – a total of 1 µL will be added for each sample. This will be demonstrated by the supervisors if necessary, as 1 µL is a very small volume, that isn’t easily visible by eye in the pipette tip. Why do we add samples in duplicate?

7) Repeat this step with the positive control in duplicate. For the negative control wells, add 1 µL of nucleotide-free water, in duplicate. What is the purpose of adding water only to your negative controls? Remember we are trying to assess the levels of all four microorganisms in your samples, therefore you need to add your sample to all four microorganisms mastermixes e.g., student 1 will add their sample 1 to every well in column 1, sample 2 to every well in column 2 etc… ensure that you change pipette tips between wells. Why is changing pipette tips between samples important?

8) When complete, plates will be centrifuged and loaded into the qPCR machine. The following thermal profile will be used for the reaction; 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 3 seconds followed by 55°C for 30 seconds. One of the teaching staff will help each group load their own plate and explain the principles of the qPCR machine, and what the results will look like. We will show you examples of a standard curve that has previously been generated, and will be used for you to quantify the level of microorganisms in the sample.