Tag Archives: UCL Institute of Ophthalmology

Glial Cell Research by Farzana Miah

Farzana learnt about glial cell research during her two-week placement under the supervision of Angshu Angbohang, in the group of Professor Astrid Limb.

During my two weeks at the UCL Institute of Ophthalmology I’ve learnt about the human glial cell, a type of stem cell, that is able to regenerate into retinal neurons.  This is important because the retinal neurons are photoreceptors at the back of the human retina which enables you to sense light and therefore allows you to see.

Scientists have discovered that the Muller glial cells in zebrafish have the ability to regenerate the retina neurons multiple times once they are damaged. This ultimately intrigues us to find out why and how they are able to do that and if one day a human may be able to do the same.These are the steps that I have taken during my placement, they all link together therefore it was important to do each step carefully as any mistake could of had an effect onto the final result:

Farzana 1

Cell culture

Firstly I saw two Petri dishes that had cells inside of them, however one dish was confluent and the other dish had cells that were sparsely separated. We used the confluent dish which had to be vacuumed as there was solution around the cells, this gave us the cells we needed and nothing else. Furthermore, the cells were attached to the dish, due to this we had to use a solution called triple E as this helped detach the cells from the surface of the Petri dish. We then placed the dish into an incubator at 37 degrees Celsius which was followed by centrifuging the cells. Five minutes later the cells were at the bottom of the dish, we then removed the triple E by aspirating it and adding 5ml of medium (which provide nutrients to the cell). Finally we stained the cells using methyl blue.

RNA Extraction

We then extracted RNA from the cells we cultured. I removed medium first and then added the triple E solution as this helps detach the cells from the dish. We then centrifuged the cells which left a cell plate present (a white disk at the bottom). We then removed the triple E solution to give us the cells that we needed to extract the RNA.

We added lysis buffer (one micro litre) and beta-mercaptoethanol (a corrosive alcohol), we mixed them together and added 350 micro litres into control and treatment tubes. After that we used a syringe to help move the solution up and down, to help the breakdown with the lysis buffer to go faster. I centrifuged the cells again and then transfered the solution into an eppendorf tube.

Reverse Transcription

Reverse transcription is when the RNA turns into cDNA by using mRNA, primers, DTT and a reverse transcriptase enzyme (such as “Superscript”)

PCR

The polymerase chain reaction (PCR) is to help the cDNA to multiply rapidly and the forward and reverse primers bind to each strand. We needed cDNA, primer, water and Gotaq (a green solution that contained everything). We use cDNA because we know how long it lasts, however a normal DNA can affect the results.

Farzana 2The primers use to test for photoreceptor are:

1) NR2E3

2) Recoverin

3) IRBP

4) Betactin

Farzana 3
Gel Electrophoresis

This was done because we needed space for the reverse transcription solution (mRNA, primer, DTT and superscript). To do this we need agarose gel, solution buffer and red gel (something that allows you to see the band).

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Analysis

The treated human Muller glial stem cells photoreceptors are more expressed than the controlled ones.
The treated human Muller glial stem cells photoreceptors are more expressed than the controlled ones.

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Polymerase Chain Reaction

Continued from her previous post, Lucky tells us about her experience with using a fundamental technique in molecular biology – the polymerase chain reaction (PCR)…

During my placement I had the opportunity to observe the isolation of RNA and the process of reverse transcription to get cDNA; with this I learnt how enzymes, for example reverse transcriptase, can be used by scientists to achieve a desired outcome. I was able to take part in PCR where a small sample of DNA is amplified in three main steps: denaturation, annealing and elongation. The samples, including my own, were run through a gel and imaged. My sample appeared as a very bold band which is good.

pcr gel

By Lucky G

Rods and Cones – a placement at the UCL Institute of Ophthalmology

Lucky recently undertook a placement at UCL’s Institute of Ophthalmology. Here she explains a little of what she learnt about vision…

The retina is the part of the eye that senses light. It consists of two types of photoreceptor cells: rods and cones. Rod cells work in low light levels, and as their name implies, they are rod shaped. This type of photoreceptor cell is responsible for what we would call night vision; they allow us to see in the dark. Rods use a chemical called rhodopsin to absorb photons. Rhodopsin molecules split into a retinal and an opsin molecule when they absorb photons. Rhodopsin then reforms from these molecules at a constant rate but this process is very slow. This is the reason why our eyes take time to adjust to the dark. When the light is on and there are high light levels, rhodopsin molecules are broken down so when the light is switched off and there is less intense light we can’t really see until rhodopsin starts to reform. Gradually our night vision, in a sense, activates. Rod cells are unable to perceive colour, unlike cone cells and so our night vision is black and white.

Cone cells are responsible for our ability to perceive colour. The cone cells of our retina function at higher light levels than rods and have a cone-like shape. There are three types of cone cells, all of which respond to different wavelengths. S-cones respond to short wavelengths and peak at a bluish colour; M-cones to medium wavelengths and peak at green; L-cones to long wavelengths and peak at red.

Our colour vision or perception of colour is based on the wavelengths of light that objects reflect. Take the example of a plant leaf; the cells of a plant leaf contain the green pigment chlorophyll. As a result, plant leaves are green and so absorb other colours of the spectrum such as blue and red, reflecting green visible light into our colour perceiving cone cells. Green light has a medium range wavelength so it is responded to by M-cone cells. Your brain then interprets signals it receives from your cone cells. People without all three cone types functioning correctly have colour blindness. So, if a person’s L-cone isn’t functioning properly they won’t be able to see the colour red properly as L-cones respond to long wavelengths like red light.

By Lucky G