minimum information for publication of Microarray

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minimum information for publication of Microarray

Postby memari » May 24 2012 7:40 pm

Is there any minimum information for publication of Microarray like this one below for qPCR???

The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

http://www.ncbi.nlm.nih.gov/pubmed/19246619

My supervisor asked me to do some QPCR to show a over expression of a gene that he has seen during Micoarray that one of his PhD student has done.

I have repeated it but there is no over expression. But in Microarray there is a 40 folds over expression between treated and non treated cells.

But my supervisor insists that I should repeat qpcr until the result is like microarray.

The PhD student is in third year of PhD and I am in the first year, so they think that my results are not trustable.

I have read in some papers that before publishing microarray data, we should verify data by RNase Protection Assay and QPCR.

What is your idea?

I have told them that maybe the cells were contaminated with Mycoplasma. Mycoplasma is a real problem:

http://babakmemari.wordpress.com/2012/0 ... croarrays/

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Re: minimum information for publication of Microarray

Postby procopio f.a. » May 25 2012 10:39 am

hello
i made many microarray validation and in my opinion your boss is rigth/wrong.

First we perform microarray validation because the rna pre amplification steps for microarray samples preparation is not very linear. So you cannot trust the values that came from a array data. If it was the case no qpcr should be necessary (like in the sequencing case).

In my experience validating microarray i have found that normally 85% of the gene by qpcr/array go in the same direction also if with different fold changes. 10% show no difference in qpcr also if by genearray you found it. A 5% of the case qpcr give the exact contrary result of the microarray.
Ofc those numbers are just indicative, sometimes we get a lot better then this.

So it can be the case that you microaray is wrong, and same for your boss.

Ofc your boss is right when ask you to continue until you don t get the result.....or you don t clearly show that he is wrong

First at all, when you talk about treated and not treated cells what you mean??? you should do a lot of attention in that. For example let confront resting t cells versus pre activated t cells. Let perform gene array and qprc on whatver gene do you want. The results????? totally wrong. why? simple the pre activated t cells have tons of house keeping genes compared to the resting one, so u will over estimate the amount of your targets in resting cell. I dunno what treated or not treated means in your experiment, but maybe your results can be driven by this matter, also because in micro array you can use many housekeeping genes to normalize or even a virtual housekeeping gene that is a bioinformatic summary of all the housekeeping genes present in the chip.

Another important facto is to make sure that the cell/treatment used for the cells for your microarray are the same for your pcr. Difference in treatments/cells can ofc driven different results.

Other inputs, did you ask to the lab that perform the microarray is they have still some not treated RNA and some amplified one ???? maybe you can perform your pcr directly there.

Last input. Are u sure about your pcr assay???? do u have positive controls, negative ones? do u use what taqman or sybeer? (the first one give less background) did you put the qpcr product on a gel???

I hope all this can help

francesco
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Re: minimum information for publication of Microarray

Postby memari » May 28 2012 9:43 pm

procopio f.a. wrote:hello
In my experience validating microarray i have found that normally 85% of the gene by qpcr/array go in the same direction also if with different fold changes. 10% show no difference in qpcr also if by genearray you found it. A 5% of the case qpcr give the exact contrary result of the microarray.


Thank you. this part of your answer was very interesting.

I can not reveal information but this is information of micro-array for one of the gene which has over-expression.

4.514------ 0.000------ 46.487-----0.000------80.337-----0.000
4.514 is for Control cells.
46.487 is for treated cells with one compound.
80.337 is for treated cells with two compounds.
0.000 are P-value.

we use evagreen for qpcr.


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Re: minimum information for publication of Microarray

Postby procopio f.a. » May 30 2012 10:26 am

evagreen..... people love it... did u put your pcr product in a gel???? maybe u are getting some non specific amplification and the evagreen will give u a signal about as well
Not affiliated with any company. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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