Friday, November 21, 2014

Stipend story of a scientist!

Hi readers,

It feels so good to write here, after a long (so long, in fact) gap. How are you all doing? I’m doing great, did you know, I have now joined M.Tech (Biotechnology) in Anna University, Chennai. And, after 20 years of life under the warmth of my parent’s wings, I have finally managed to spread my own wings, out of home. Yes, I have come out of my kutty (small) town, to a big city – Chennai. It feels good in one way but the other? No! It’s not feeling so good the other way. After coming here, I have realized that I was away from the so much polluted world for 20 years. This place, because of tremendous industrial development and stuffs, is polluted extremely, that you should cover your head, face, hands, foot, literally, all your body parts except eyes to keep you clean and safe from the poisonous, dusty, dirty air! Ah, forgot, you should wear big coolers to protect your eyes too. Sometimes, it feels like there is not more than 10% of O2 in the air! Villages, towns – Rural areas are heavens to live in, but, I came running to this city to achieve something; to breathe some dirt; to experience how it feels really to be independent and motivated towards a goal; and, to go crazy; to study like I have never did; to feel, try and learn everything under the sky; to make a new episode of my life!

It’s very challenging in India, especially for a girl from a small town, for a girl from an orthodox background to get out of home and go behind her dreams. After facing, so many distractions, “get married” advice, I finally convinced my parents that I would like to get out of my town, away from their warmth, avoiding their spoon feeds. I proudly was explaining them that how I cleared the entrance and how much stipend amount I’ll be getting every month, so that, they need not take care of my expenditure anymore! I was building so many beautiful castles in air with the thought of getting stipend and spending it for my food, shelter, clothing, books and if possible a small tour. Wondering, how much is the stipend money? It is just 8000 INR :P and it WILL NOT be sufficient even for basic needs if I’m spending it, without being a little stingy!  And, yes, I was planning so much with that 8000 INR.

Do science!
At last, convinced my parents, came here, joined. And, the best part of the story starts here. It’s been 3months since I joined M.Tech, and, I had not received stipend, even for a single month! So sad, right? First month end, I asked my dad, “money”. He dint ask about the stipend, but, I felt so much embarrassed and started explaining him that I would get 16k altogether, at the end of next month. He said, “It’s fine, even if you don’t get it!” But, it dint feel good for me!

When we (all poor students of my class) asked about the stipend in the university department, they said that they dint receive the funds yet. I don’t know, what the problem IS, but, it is JUST not right! Every other student in my class, having left their home town, having travelled so long, from all the corners of the country, are just SO MUCH worried because of this. We know that we would receive the stipend money for all the months, for the whole years, totally around 2lacs, for sure, without fail, but the question is, “WHEN?” The stipend is meant for supporting the student’s expenditure, but, what is the idea of giving it after a long… long… gap? It won’t be of any use if we receive it, after completing M.Tech, after finding a job! We need it now; we need it to take care of ourselves and to proceed with our research. And, I seriously understood the reality why every Indian student wants to fly abroad for doing MS. At least, they would get the stipend on time, there!

Money!!! Stipend! :(
If this is the case, YOU people, who are in charge of this stipend amount, don’t announce or promise that we would receive a monthly stipend! I know, writing here, will not move a brick, but, still, just to vent my frustration out!

Sorry, for venting out all the dirt over you. You researcher, just  GO, run your gels, heat up your PCR (I don't know why, but I really, love this guy) machine, work, research, toil, not to receive anything out of it, but satisfaction of going behind your dream. After all, I’m happy that I’m doing what I love! You too?  Yaai, Cheers to us! Got a doubt? About the "scientist" mentioned in the title," It's me :P"

Yes, lets do it!
But, look around, life is so much beautiful with big red roses all around and very little, less thorns! What do you say?

Thursday, January 16, 2014

Real Time PCR (qPCR)

I don't know why, but, I really love this guy! :P Yes, he is PCR! You ask me anything, like, what technique we can use for introducing mutation? what for diagnosis? I'll answer PCR for all! :P Let me talk about him for sometime here.

PCR (Polymerase Chain Reaction) is used for amplifying any gene from a given sample using Taq polymerase, dNTPs, Primers and buffers. After PCR the amplified product or amplification is generally checked by running an Agarose gel electrophoresis. But, in case of Real Time PCR there is no need for running gels as the progress of the PCR is monitored online with the help of fluorophores. Generally SYBR Green dye, Taqman or molecular beacon probes are used for Real Time PCR. In SYBR Green method, the fluorophore binds with the double stranded DNA and produces fluorescence, and hence as the amplification increases, fluorescence increases, but the fluorophore has no specificity and hence, even if the amplified product is not the product of your interest you would find increase in fluorescence. The TaqMan and molecular beacons are probes which bind which are labeled with fluorescent dyes at one end and quencher at other end. When the fluorescent dye and the quencher are at close proximity there won’t be any fluorescence but on amplification the fluorescent dye and the quencher gets apart thus causing increase in fluorescence. These are specific to the gene amplified and hence we could monitor the amplification specifically online.

Another method is LUX (Light upon eXtension) in which the primer is labeled with fluorophore at the 3’end. The primer remains in a hair pin like structure initially, and, at that configuration it emits less fluorescence. When the primer becomes linear single strand, the fluorescence increases and when it gets extended and becomes double strand, the fluorescence increases further. As the primers are specific, the fluorescence increases only when the amplification is specific.

Melting curve analysis is generally done for the PCR products in case of LUX and SYBR Green as the fluorescence could also be due to the primer dimer. In the melting curve analysis, the product is heated thus allowing it to unwind, the melting point temperature (Tm time taken for half of the sample to unwind as single strand) is noted. The melting temperature will be different for primer dimers and the product as they differ in length. And hence, fluorescence formed could be exactly correlated only with the product after this analysis. Many of the light cyclers have inbuilt option for performing this melting curve analysis.

It has various applications; few of them are listed below:

  • Diagnosis where genes which are specific for a particular species can be targeted and amplified, if the amplification is positive then the sample could be diagnosed as infected. The specialty of qPCR is that it also gives exact severity of infection.
  • Microbiology – for comparing the differences between different species, sub species and serovars of microbes
  • Research  - for works like cloning amplification of a particular gene is essential and also for measuring expression of a particular gene qPCR can be used.
Hope, you also loved him! ;) 

Tuesday, January 14, 2014

Knock Out Mouse (KO Mouse)

Knock Out mouse is a genetically modified mouse in which a particular gene is removed or inactivated. As a particular gene (which encodes for a particular protein) is inactivated, the mouse shows changes in external features or physical and biochemical characteristics. The first recorded knock out mice was produced in 1989 for which Martin Evans and Oliver Smithies were awarded Nobel Prize.

The various reasons for producing knock out mice are

·        To understand the function of a gene
·        To understand various diseases which are due to alterations or mutations in genes
·        It gives an idea for understanding and treating various diseases
·        To develop and test various drugs produced

Some of the diseases which are widely studied using mouse models are neuronal diseases like Parkinson’s disease, Alzheimer’s disease and various types of cancer are also understood with the help of KO mouse models. Besides these, KO mice are also used for studying heart diseases, diabetes, obesity etc.

For producing knock out mouse, a DNA fragment is designed such that it contains some random sequence in place of the gene to be knocked out with flanking sequences similar to that of the sequences which are flanking the particular gene (to be knocked out) in the original genome. Moreover, the DNA fragment also contains markers to detect the effect of knock out or Embryonic stem (ES) cells are generally used for knock out, the ES cells are collected and transfected with the DNA fragment designed. Homologous recombination occurs resulting in knock out and the cells which are knocked out are transferred back into the blastocyst, then the blastocyst is placed in the uterus of a surrogate mother (mouse) which gives birth to knock out mice.

Why knock out “mouse”?

The next question that comes into our mind is “why mouse? Why not other species?”
The reasons for using mouse as a model for understanding human diseases and genes are the similarity between the genome of mouse and humans to a greater extent. And also mouse is easy to handle.

There are also limitations in producing knock out mouse. Most of the knock outs are lethal so the knocked out mouse cannot grow up and survive for a longer period. It is difficult to produce a knock out mouse as there are always possibilities for random or non homologous recombination. Producing knock out rat is difficult, and the first knock out rat was produced in 2003, 14 years after the production of KO mouse.