Day 13

Day 13 - Let the project begin

When Zhen Dong came to the lab in the morning, he asked me where were the embryos. I told them I only had 10 (which were the remaining embryos that I had not given to Shen Qian and Foong Mei) and he sort of scolded me. The previous day he told me to collect at least 20. Then, I told him that I had isolate some RNA from lots of embryo yesterday. So I just pray that my RNA had a decent quality.

I waited for Shen Qian and Foong Mei before I ran my gel to see the RNA bands. And once again, I made a mistake. I loaded DNA ladder instead of RNA ladder but fortunately, the result was good. At least I got 2 clear band and 1 fuzzy band. the 2 clear band corresponded to rRNA while the fuzzy band at the lower part of the gel correspond to the tRNA. The smear at the band was the mRNA.

After that, we proceed with reverse transcription-PCR (RT-PCR) to isolate and amplify the desired mRNA. In RT-PCR, complementary DNA (cDNA) was produced using RNA as a template, therefore the name Reverse Transcription. After RT-PCR, we performed PCR using the primer that Zhen Dong and I have designed (the sample) and Beta-actin primer as our positive control. We left our PCR in the machine overnight as we were short of time.

Day 12 - the Hairy Roes

Day 12 - the Hairy Roes

That day I finally had a very vague idea of what my UROPS project is about

I am doing a research on a gene called VOX and to be honest, I don't have any idea of that gene except that the gene is expressed on stage 13 onwards in the fish embryo. Zhen Dong promised to send me a paper regarding VOX gene but until now I have not received it.

The first thing I have to do was to confirm the sequence of the VOX gene and to do that, there are multiple steps involved. The very first thing I did was to select the Medaka embryo which were already at stage 13 and above.

Medaka roes are very different from Zebrafish roes. While zebrafish's are smooth, medaka roes are hairy. The roes are clustered together with a very fine strands of brown fibre. Although the fibres were very fine and thin, they were very strong. The fastest way to separate them is to wind them around a pair of needle.

After selecting the embryos, I proceed to do a RNA isolation. I did not know what was wrong with me that day, maybe I was just to nervous to start my project, that I made lots of mistake. I forgot to aliquot the trizol reagent to smaller container and I even dropped my tip inside the bottle. I could sense that Yanyan who were there at that time was thinking that I was just a nuisance in the lab.

Foong Mei and Shen Qian had nothing to do so they wanted to join me in my experiment. I gave them some of my fish embryos. I guessed they were too lazy to select the embryo as they just took a bunch of them to the tube.

I did not finish my RNA isolation so I stopped at the washing steps.

Day 4 - Butches among fish?

Day 4 - Butches among fish?

My mentor told me that we couldn't do Western Blot today because we could not get fish embryo to do it. He just asked me to follow whatever Shen Qian and Hong Ni were doing. So I went to their bench to join them.

The schedule for that day was to do RNA isolation and Genotyping the fishes. The purpose of Genotyping is to determined whether the fish is genotypically male. The difference between female and male fish in medaka is at their back fin. Male medakas have discontinuous jagged back fins whereas their female counterparts have continuous smooth back fin. However, not all medaka with discontinuous jagged back fins are males, some of them are genotypically females (so I called them butches, female with male appearance).

After preparing our RNA sample from medaka's ovary and ran them for RT-PCR, we went to the aquariums to get some supposedly-male medakas to be genotyped. What we did was to cut a small part of their tails, homogenize it to extract the DNA, perform PCR and run gel electrophoresis. If the fish is genotypically male, two bands will be observed from the gel run, but if the fish is actually a female fish, only one band will be observed. We took samples from 34 fishes and put them back in separated aquarium.

After that, we went back to our lab to homogenize the sample using a pestle. The pestle really looks like a large size cotton bud. It is white in colour and has two bulging ends. Crushing 34 small pieces of fish tails were quite tiring, luckily we split our jobs. After we homogenized the sample, we added celects, a reagent that will bind to everything that will interfere with PCR. Then we performed PCR to each sample.

While waiting for PCR to finish, we run a gel to visualize our RNA isolation sample. The result was very disappointing as I only can see a smear on my lane showing that my RNA has been completely degraded. Most probably I was not careful enough while handling the reagents... Sigh....

In the end, we decided to run our gel for genotyping on the next day as it was getting late.