Getting funded is not the same as succeeding (Seth Godin)

One of the reason I can think of when I wrote the blog entry entitled Scientist falsified research data...... on the reasons why some scientists will falsified research data (in my opinion) is to get more research grant.

However, Seth Godin has a very neat opinion regarding getting funded and be successful.  In his opinion, getting funded is not the same as succeeding.  Totally agree on this.

In his blog, he mentioned about this "I don't care so much how much money you raised, or who you raised it from. I care a lot about who your customers are and why (or if) they're happy."

And I hope he don't mind when I try to change part of the statement to make it more relevant to comment on Philippe Bois's act to falsified data in two published papers, "I don't care so much how much money you raised, or who you raised it from. I care a lot about how genuine your published data is, which has been validated, before I consider how useful the published data to the society".

Think about it.

Scientist falsified research data......

The act of Philippe Bois to falsified data in two published papers has caused him to be banned from participating in federally funded research projects for 3 years.

This news suddenly made me remembered of  The SPEED of Trust: The One Thing That Changes Everything which I read few years ago.

The news for the act of distrust also travel quite fast though. 

I always can't imagine why there is a scientist who wish to falsified the data.  One the top of that, I really can't think of a solid reason for his act.  To me, publication in science is always been viewed as knowledge sharing.

Is it due to the following reason:

• To get more research grant,
• To get his job secured, or
• To get promoted as soon as possible by achieving KPI set by the institute?

Not sure if there first case was done by Hwang Woo-suk in Korean Stem Cell Scandal?

I guess, currently there is a sad shift in the purpose for scientists to do their research and get their finding (s) published, and I hope scientific committee should be aware of it and take it seriously.

Exponent negative equals to negative result in real time PCR

22 June, 2011, I went to meet a customer complaining that their real time PCR results were not consistent over the week, where the positive result in one run becames negative in the next run, with the same sample (s).

Before I went, I got some run files from her.

Back in the office, I noticed that there was nothing wrong with the result (s), except that there are some minor differences in her replicates, which could be pipetting error.

To my surprise, after having some discussion with her on how to analyze her result (s), she told me that the way she call a sample as positive or negative based on the exponent from result in the Result column generated by the software; i.e, 2.223e+1 is positive, whereas 2.223e-0.1 is negative.

OMG, she is analyzed her result based on the exponents of the quantitative results generated based on the standard curve she had.

I ended out discussing with her what is the meaning of exponent, and how to interpret the result.

Wow, this is the most creative way of analysis a real time PCR result so far I met.

salary in research......

The figure below is from http://www.genomeweb.com/sites/default/files/newspics/genometechnology/Median%20Salary%20by%20Region.jpg

So, does the relatively low income for researcher (s) in Asia/Pacific region does explain the relatively low excellence discovery and research finding (s) in this region?

personalized medicine......

Some of the shocking statistic I found from the http://the-scientist.com/ on personalized medicine as follows:

• FDA recommends that doctors genotype patients before precribing more than 70 commonly-sed medication for specific genetic biomarkers
• Although 98% of the physicians agrees that genotype their patients might be useful to determine their drug response, only 10% believed they were adequately informed about how to test their patients for biomarkers that may predict the safety and/or efficacy of a particular drug, and
• New biomarkers are identified each day

Question (s) that flashes to my mind now (at the point of writing this entry)

• What is the percentage of patients we have so far gains benefits from personalized medicine based on genotyping?
• How long does it normally take for the information to transfers from research laboratory (s) to clinical laboratory (s)? And how long again does the physician will take to really appreciate the newly discovered biomarker (s) or assay?
• And, how many patient (s) who is really aware and confident of the benefits of personalized medicine?
• Of course not to mention of how many percent of physicians can truely and effectively explain the concept (s), benefit (s) and importance of personalized medicine in a way that the patients can truly understand?

company's propriety issue?

Recently, in order to reduce the time and effort to design and optimized an real-time PCR or qPCR assay, lots of scientist opts for pre-designed assay from certain company (s).

These assays are indeed helps a lot of scientist by reducing their time and effort to design their own assay where they need to consider the primer (s) and/ or probe (s) specificity and efficiency, mispriming and etc.

However, one of the common issues faced by the scientist when they wish to publish their data using these assays is due to the company's propriety issue, the sequence information for the primer (s) and probe (s) can not be disclosed. But these information is critical as it is required by MIQE guidelines.

So now, does these companies still want to keep these information to themselves, which might affect the sales of these assay (in my opinion) or just do the customer a favor by disclosing these information so that there are more citation for these assay in near future??????

not statistical significant......

Been asked this question this question a lot of times before, "Why my result (s) is not statistical significant though the differences between the control and treated groups are big???"

Please have a look at the standard variation of your data, and most likely that the standard variation for one or all of your experimental group (s) is huge enough to cause your result is not significant though the mean difference between your experimental groups is big.

Try to think of ways to improve your experiment's data accuracy and precision......

sterilization

Wondering is there any method to completely made an item, device or solution to become completely free of all living microbes and viruses? I guess nope, as far as I know of......

Is it sufficient to reach to the level of the probability of a microorganism to survice on an item subjected to treatment is one in one million to be called as sterile?

Biosafety......

Currently preparing some slides for a presentation on lab biosafety.......

In the midst of preparation..... suddenly these questions comes to my mind.....

"We can do so much on ensuring ourselves are safe from the contagious microorganism in lab (s), be it clinical, diagnostic or research lab (s), wondering how many of us are really thoroughly wash our hand after using the toilet every time???"

"Wondering if you have any idea of how many pathogen (s) is there on our hands after each session of toilet visit?"

reuse spin column.....DNA....and RNA

Come across another publication on the way to reuse DNA purification spin column......

Complete decontamination and regeneration of DNA purification silica columns

And for RNA spin column as well......

Regeneration of total RNA purification silica-based columns

The question is, should we give it a try??????

Or perhaps, we can think of this as a more environmental friendly way for doing research???

Spectrophotometer....DNA and RNA

Can a spectrophotometer reads at A260 differentiate between DNA and RNA samples?

The answer is of course not.

pyrosequencing and PCR

I made a mistake by answering "Yes" to this question, and probably I will say "No" if I have think twice to it.

The question is "When you run the agarose gel, you get only a single sharp band; does this means that your pyrosequencing will be fine?"

OMG, I shouldn't have answered "Yes", but should be a firm "No" or just a more polite "Probably".

The issue here is, before subjecting a DNA sample for pyrosequencing, it needs to be denatured into single-stranded DNA after the PCR. And to get the biotinylated PCR product to become a single-stranded DNA, we need to captured or immobilized the biotinylated PCR product to sepharose beads, which will be then denatured and washed, and finally been released into a plate which contains the buffer that has all the other component (s) for pyrosequencing reaction.

Of course, without a good PCR product, you will not get any pyrosequencing result, but by having a good PCR product will not promise you to have a great pyrosequencing product or result as well, because as you can see, there are so many steps are taken place after PCR which could go wrong if you are not careful.

So, the answer to the question "When you run the agarose gel, you get only a single sharp band; does this means that your pyrosequencing will be fine?" should always be a firm "No".

RT-PCR and/ or qRT-PCR

What will you suspect when you get a negative expression of your gene of interest but a positive expression of your housekeeping gene? Furthermore, maybe you wish to try to get a hint if your primer set is working fine by amplifying the gene of interest with a DNA sample instead of a RNA sample, and yes, it amplifies.......

So, what could go wrong here?

• Poor RNA quality? May be. But should be checked prior to any downstream assay, if possible.
• Not optimized primer design? Could be.
• Not optimized RT-PCR or qRT-PCR condition? Perhaps. I still remember when I used to amplify low abundant gene (s), and just by increasing the number of cycle from 40 to 50 cycles, all the genes got amplified.

Or the gene is not expressed in the sample then...... Worth to get this investigated, I guess, in my opinion.

denatured alcohol???

Please don't ever try to use absulute ethanol which is less than 96% to 100%. In an 95% ethanol or worse still the denatured ethanol, it contains at least 5% of impurities such as methanol, butane and isopropanol which might not evaporate efficienctly on dry spin column.

This is critical as ethanol is needed to wash away the salt used in the purification process, and imagine what will happen if the spin column does not dried completely prior to the elution steps..... Lot's of salt and other chemical that are present in the reagent (s) used in previous step of extraction and purification. After all, what is the point of performing a washing step prior to elution if most of the salt (s) or regent (s) can't be effectively been washed away???

And perhaps you might wish to list out what is the potential salt (s) or reagent (s) that could be carried over, which will detrimentally affecting your downstream assay?

A quick check, perhaps you can have a look at the A260/ A230 ratio of the purified nucleic acids that you have.

Reuse spin column......Perhaps it is something to think about......

Come across this publication, "Regeneration of commercial nucleic acid extraction columns without the risk of carryover contamination" published at Biotechniques......

I should have come across this few years back when I did used a lot of spin column for my research..... and at least I should have tried the method myself when I wrote this here today......

Giving some thought (s) after reading this publication, wondering how many people had actually tried this out? And how successful and how reproducible is the method outlined?

And will you be taking the risk by re-using the spin column to reduce the cost over the precious sample (s)???

A260/A230 ratio

A260/A230 ratio is such an essential ratio to check the purity of nucleic acids besides A260/A280, which is more commonly used and better know, but it is not widely been used.

Wondering, besides protein contamination, do you think that salt (s) or other reagent (s) contamination, which most likely coming from the carry over during the extraction and purification step (s) will actually affect detrimentally to your downstream assay (s)?

If yes, perhaps you should put slightly more attention to A260/230 ratio then......

a better data analysis method???

Lots of people will be more concern in data analysis of their qRT-PCR result and will straight away ask me which data analysis method is better than the other so that they can use for their studies. I encountered this question (s) a lot during the installation and training of real time PCR.

However, in most of the case, I found out that they don't even know of or consider the essential of starting material/ sample quality, how to process their samples to get a pure and intact nucleic acids, the quality of their extracted and purified nucleic acids etc etc. These could detrimentally effect the reliability and quality of their data.

And of course, not to mention on the experimental design. Another area which lots of people or researcher (s) overlook its important.

troubleshooting guide.....

As you go down the list of possible cause (s) explaining the issue (s) you faced, wondering do you notice that at least more than 50% of the possible cause (s) of the problem (s) you faced could be due to your mistake (s)?

With this, will you ask yourself this question, "Am I did something wrong or did I overlook some essential steps just now?" next time when you face any issue?

lyophilized primers, concentration.....

Normally primers for PCR are shipped in lyophilized format, and prior to use, these primers need to be dissolved in TE buffer to provide a stock solution concentration of 100 µM.

Wondering have you ever measured the concentration of primers' stock solution with spectrophotometer???

If not, how sure are you that the concentration is accurate???

lot number??? expiry date??? storage condition???

How many time have you check on storage condition, expirary date and/ or lot number of the specific kit (s), or reagent (s) when you received them, before you store them and/ or when you are about to use them?

Are they important? Oh ya, if not, why these informations are printed on the kit or reagent you have purchased?

Perhaps you will be more appreciate them when you are in the midst of troubleshooting your assay as you are going through the long list of the troubleshooting guide......, maybe, just my opinion. =)