NormFinder for analyzing stable reference genes

NormFinder is another tool (an Excel add-in) to validate candidate reference genes. It best works with MS Office 2003 and up. Like GeNorm, the user has to convert the raw Cq values to relative quantities by using the ∆ct equation as shown in the last post on GeNorm. I have used NormFinder with MS Office 2003 on Windows 10 system. NormFinder can also be used with R, but I haven’t tried it yet.

Once the data is prepared i.e. raw Cq values have been converted to relative quantities, follow the steps mentioned below:

  1. Once you have calculated the relative amounts, arrange your data like this. The reference genes should be in columns, and the samples (including the control(s)) should be in rows. Here 1,2,3…. are the sample names while A, B, C…. are the gene names. Don’t forget to add “group” at the end. Grouping enables to show stable reference genes in groups. Here I have two different time points i.e. 1-5 samples are from the 1st time point, and 6-10 are from the 2nd time point. We will come to it in the next steps.1
  2. Now you are ready to start NormFinder. Once you double-click the add-in file (downloaded from the site), it should show in the menu bar.


3. A short disclaimer message will show up. Just click Ok.

Screen Shot 2016-08-26 at 11.02.04

4. Select the input data just by dragging all the cells including the group.

Screen Shot 2016-08-26 at 11.02.34

5. Check all the boxes – Sample name, gene name and group identifiers. If you don’t include the group identifiers, then uncheck it. But as I suggested earlier, it would be useful to have the group identifiers.
Screen Shot 2016-08-26 at 11.02.25

6. Click on Go and within a few milliseconds it will create a new tab and would show            the results like this:
Screen Shot 2016-08-26 at 11.02.48

7. If you uncheck simple output only, then it will show a detailed result page which includes stable genes, Intra- and inter- group variations. But if you check simple output only it will show only the ranking of stable genes and the most stable genes. The advantage of using group identifier is that it gives a combination of two best genes which can be used. For example, in the above case, we can use only gene A or we can use both gene A and J as the most stable gene combination.

Screen Shot 2016-08-26 at 11.03.10

1. Normalization of Real-Time Quantitative Reverse Transcription-PCR Data. A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. 2004, Andersen CL et al.
2. NormFinder Documentation.
3. NormFinder plugin: – Check the download section on the right-hand side.

Epilepsia citation style

I was submitting a paper to Epilepsia and I use “Paper” as my citation manager (for more details, please see the “Scientific tools section”). Everyone has their own citation manager. Some use Endnote which is popular but a paid one while others prefer using Mendeley which is an open source program. Yes, you heard it right, it’s free !! And then there is Papers which is not free but is cheaper compared to Endnote.

Coming to business, while submitting to Epilepsia I found that they have a new reference style which is a modified version of “Vancouver superscript style”. The only changes are after three authors, “et al.” should follow, no need for entering the month of publication, and the use of journal abbreviation instead of the complete journal name.

I started to make the changes manually as there is no citation style that matches and the patch that Epilepsia provides are “.OS style patch” which works only with Endnote while Papers, Endnote, Zotero only accepts a “.CSL style”. Therefore, I modified the Vancouver style patch to make it compatible with Epilepsia using the visual editor. The only problem with the new “Epilepsia citation style” is that it doesn’t recognize many journal abbreviations (out of my 40 references, it recognized around 25 and changed it) so you have to do it manually. Other than that, it works perfectly. I am not a pro at Visual Editor so if anyone finds a way around to get this fixed, please let me know.

Just download and drag the file to your reference manager (works for papers) and it will install it.

You can find more citation style which is not available on your citation managers.

Hope it works for everyone else too. If you encounter any problem, let me know and I will try to fix it.



Guidelines for qPCR

RNA extraction:
1. While extracting RNA be careful on the use of Trizol. Trizol inhibits microRNA (Trizol skews microRNA results). There are two kits which are trizol free – Zymoresearch RNA extraction kit and Qiagen miRNA isolation kit.
2. To avoid frothing of tissue, while homogenizing, I would recommend using “Reagent DX” from “Qiagen.”
3. If the kit contains in-column DNase treatment, then you should use it – saves time.
4. RNA elution could be done in either DEPC water or TE buffer and store at -80°C. If possible, RNA should be aliquot and stored so as to avoid repeated freeze-thaw.
5. RNA should be measured (if thawed again) for determining if there is any degradation. Before measuring RNA, give it a brief spin and pipette it up and down at least thrice so that the exact measurement of RNA can be determined.

cDNA synthesis:
1. cDNA should be made immediately or within a day of RNA extraction so as to avoid freeze-thaw of RNA.
2. After reverse transcription of RNA, dilute and aliquot the cDNA.
3. Store all the aliquots at -20°C and take out one at a time. You can store one aliquot at 4°C for a maximum of 1-2 months.

You have to be extremely careful while preparing the plate and the qPCR machine.
1. Wipe off the bench and all the pipettes that are going to be used for qPCR first with 100% ethanol and then RNAse Zap. You can purchase RNAse ZAP from Sigma-Aldrich, which is cheaper (link below) compared to life technologies.
2. First, make the master mix by adding the qPCR master mix, the primer pair, and water. After, you are done with the primer pair place it back in the fridge or keep it far away.
3. Now take out the qPCR plate from its package and pipette the master mix to the qPCR plate.
4. Take the cDNA out of the fridge and add it to the qPCR plate and immediately cover the plate with optical seal cover.
5. Give the qPCR plate a brief spin down (high speed for 2 min).
6. Place the qPCR plate in the machine and run the program.

Links to products:
I am in no way endorsing the products, be careful with your samples, and I am not responsible for the loss of your valuable samples. I would also recommend the use of DEPC water. These products work best in our research

DEPC water:
Reagent DX:
Zymo Research RNA extraction kit:
cDNA synthesis kit:
qPCR master mix:
RNAse Zap:



Guidelines for publishing your article

Some of you reading this may have already published and some of you are writing your manuscript and some are going to submit. Here is a nice post published in Biomed central about how to make a good impression to the editor and get your manuscript for peer review.

The title of the article is How to get published: making a good first impression. It may help you and it has some nice tips on the “abstract title”, the “abstract” and the “cover letter” for the journal.

Things to consider when applying for PhD positions

Applying for Ph.D. positions

When you apply for positions, both Ph.D. and job positions you need to have a “heart of steel” because the rejection ratio would be much higher than the acceptance rate. For example, if you apply for 20 positions, chances are you may get invited for an interview for only 2 or 3 posts at the max. By interview, I mean for a Skype interview and after the Skype interview there is still a chance that you don’t get invited for a personal interview.


This is the scene when you apply for positions in the European Union (EU) countries. Getting a job especially in academia has become tight nowadays (It might be the case everywhere else in the world, but I’m talking from my experience). You may have all the skill set required for a position but always remember there is someone else who is applying for the same job and maybe having that extra oomph factor which will drag your application to the bottom.

There are certain things to be kept in mind while applying for positions:

  1. You should be crisp and clear while writing a mail to ask for positions or apply for positions. The professors and scientists get many emails from prospective students for positions. The subject line should be catchy. For example, a subject line like “looking for positions”, “job required” certainly doesn’t catch attention. But subject lines like “Ph.D. position: Memory Formation or Neurobiology of Nociception” catches the attention of the reader.
  1. Next comes the body of the mail. You shouldn’t have a long body which spans for more than 1 page or let’s say even a complete one page is too much. I would say be concise and have the following points:
    a) Your very brief introduction
    b) The position you intend to apply for
    c) Your qualification in brief
    d) A thank you message for the time the professor will invest reading it.

Now comes the time for the most important documents: The CV and the Motivational letter. These are the key components of your application, not to forget the importance of your grades and certificates.

Curriculum Vitae – Your CV should be well organized, in a standard font (preferably Times New Roman with the font size of 12) and should be properly aligned. For headings, you can use a slightly bigger font or make it bold. The following points should be mentioned: Education, work experience, essential lab skills, achievements, publications, and references. You should also include computer skills – this should include the computer language(s) and software you know. By software I mean not the Microsoft Office package (that everyone knows) but software like SPSS, Graphpad, Matlab and so on.


Motivational letter – This is also an integral part of your application package. This letter should explain your past achievements, present positions, future goals and why are you interested in that particular job. If you have some research questions related to the position, don’t forget to mention them. Your future goals should include “Where do you see yourself in 10 years”, “will you remain in academic research or move to industry.”

  1. While sending your application to combine all the documents in a PDF file with the CV on the top followed by the motivation letter and then attach all your diploma and certificates. Rename the file to something like “Twain_documents” or if mentioned in the advertisement how to name your file, do it accordingly.

If you send your application in this manner, there are high chances of the professors reading the same. But responding to it depends on them. They sometimes may react instantly; some may take a few days to reply, and some don’t respond at all. If you don’t get a response within a few days, it is better to send a gentle reminder to the professor about your application but don’t keep on sending reminders and bug them. If you have applied for an advertised Ph.D. position then better wait until the application deadline is over. I remember I sent a request for an open Ph.D. position but didn’t get any response for three months. I tried to make calls to the professor, but nothing helped and suddenly one day I got a reply saying that my application has been declined.

If your application has been accepted and considered then its time to get prepared for the Skype interview. Be well dressed, don’t appear for the Skype interview as if you just got up. Don’t get too excited and don’t assume that if you have been invited for a Skype interview, you have a higher chance of getting in. The Interviewer would be taking Skype interviews of more than one candidate. This meeting would be a bit informal, and the professor would like to know more about you, your plans and your goals. You may get questions like “tell me about your thesis”, “what drives you in science,” “why did you apply to my lab” and sometimes some technical questions. You should take this chance to get to know your future PI as well. You should ask questions about the project, how many students and post-docs are there, the techniques that are going to be used in the project, chances of publications, etc. This is the best time to get to know each other.


So, if you are lucky, and the professor is interested, then it’s time to face the personal interview. If you have applied for advertised Ph.D. positions, then you present your master thesis in a small group where the professor, the post-doc, and other Ph.D. students would be present and after the presentation, you would be shown around the lab and also get a chance to interact with fellow Ph.D. students and post-docs. This is your chance to get to know the lab better, if the students are happy with the personal investigator (PI), the publications of the Ph.D. students and if the PI sends you for various national and international conferences. But, if you have been selected for a Ph.D. program then you have to present your thesis in front of a selection committee and sometimes they ask you to submit a paper as well. Be well dressed, but don’t overdress yourself. Don’t wear a suit and go but also don’t wear shorts and go for the interview. A shirt with a nice pair of jeans usually works (again, as I said, in Europe). The way you present yourself, the way you dress and the way you speak are all noticed by the group. So, try to be confident and be clear on what you say.

A small mention for students who are applying to the UK for Ph.D. positions. Please note that funding is only available for UK or EU students but still you may enquire about funding possibilities for international students.

To conclude, I would like to say that don’t give up if you are rejected. Try to be positive and apply for more positions. You get to learn something from each application, and you can use the knowledge to enhance the next application. Hope this article would help students who are applying for Ph.D. positions. I wish you the best with your applications.

Some job searching sites:

  1. – A lot of positions related to neuroscience is advertised daily.
  2. – You can find a lot of employment here. Not just Ph.D. position, but commercial positions as well.
  3. – This is a good site but the positions advertised here are mostly in Germany and sometimes elsewhere. The site has an English version also. You can find industry job positions as well.
  4. – This is a social networking site for scientists, but it has a “jobs you may be interested in” column as well. You can subscribe to weekly/daily digest of posted jobs here.

If you know of any more sites, please do share them.

Hope this would help for those who are applying for Ph.D. positions, and if you have any questions, please feel free to leave a comment.