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an urgent problem with replicates


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#1 Mohamed 1984

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Posted 03 April 2014 - 01:23 PM

Hi All, 

 

Some of my colleagues designed a challenge experiment but they ignored the biological replicate. I have now lung, spleen from one H5N1 challenged mice. I want to measure the virus titer in these sample by qRT-PCR. 

 

How can I do this without need to repeat the experiment ? 

in another way, is there is any test by which I can measure the virus titer؟ 



#2 bob1

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Posted 03 April 2014 - 02:22 PM

qRT-PCR would work so long as you have some reference samples that tell you which CT corresponds to which CPEU. 

 

You could do it the old-fashioned way and titrate the virus by plaque assay.



#3 Mohamed 1984

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Posted 04 April 2014 - 12:02 PM

Thanks, 

I have refernce samples but could u explain in details how to run the qRT-PCR here. 

My sample is stored in RNA later. Is it still duable to do the plague assay 



#4 bob1

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Posted 04 April 2014 - 12:43 PM

If they are in RNAlater then there is only a very minimal chance that they virus will still be viable.

 

qRT-PCR would rely on you identifying a certain gene that would tell you how many copies of the virus particle are present and then using standard dilutions to work out CT values related to the number of copies.  I don't work with influenza, so I can't give you any more detail than that.



#5 Mohamed 1984

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Posted 05 April 2014 - 11:01 AM

I did qRT PCR several time. I understand what u told me about, but still no biological replicate. My question is how to make statistics in case you have only one biological sample ? 



#6 Trof

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Posted 06 April 2014 - 10:58 AM

You can at least do several tissue isolations from one animal.

 

But that's not a true biological replicate in such case. You can only see variations between different parts of tissue or/and isolation protocol.

 

But you just can't make more mice from a single one.


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#7 Mohamed 1984

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Posted 06 April 2014 - 03:19 PM

That is true and this my problem. Is there is any solution ?



#8 Trof

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Posted 07 April 2014 - 01:25 AM

Repeat the whole experiment with more biological replicates. Or just simply repeat it and pool both controls and affected mice (separately).

 

Alternativelly, find a necromancer that will summon back some of mice from dead..  


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#9 Mohamed 1984

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Posted 07 April 2014 - 12:59 PM

Ok, The kast solution seems the best. you could save time with answering this from the begining without need to make jokes. 


Edited by Mohamed 1984, 07 April 2014 - 01:00 PM.


#10 Trof

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Posted 08 April 2014 - 03:00 AM

Well sorry, but I gave you IMO relevant answers in every post (yes, even in the last one). 

 

But I just can't help it when you ask twice how to make more mice from one..


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#11 Mohamed 1984

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Posted 08 April 2014 - 10:21 AM

Thanks for your answer. 






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