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Which one is better as reference gene base on these data

reference gene realtime pcr qpcr

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#1 memari

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Posted 07 November 2012 - 11:27 AM

Which one is better as reference gene base on these data?
I (New student) have a debate with a PhD student (He has started his PhD three years ago) about choosing a reference gene.
I say that GAPDH is good. But he says that 18s is good.
What is your idea? 1 is non-treated cells and 2,3 and 4 are cells which have been treated differently.

---------------- First Repeat-- Second Repeat-- Third Repeat-- Average
1-- ACTIN- 14.0449068-- 14.0022355-- 13.9916405-- 14.0129276
2-- ACTIN- 14.0670855-- 14.0977196-- 13.9009442-- 14.02191643
3-- ACTIN 13.9944372-- 13.7456839-- 13.7724482-- 13.8375231
4-- ACTIN- 14.2457309-- 14.2877965-- 14.0749623-- 14.2028299
1-- 18S- 9.2736629-- 7.4010807-- 8.5428005-- 8.405848033
2-- 18S- 9.2615854-- 6.7311396-- 6.7095866-- 7.5674372
3-- 18S- 9.2900732-- 9.30192-- 8.5359895-- 9.0426609
4-- 18S- 8.5324967-- 8.5267837-- 6.9371083-- 7.998796233
1-- GAPDH- 17.3842867-- 17.5144233-- 17.2672231-- 17.38864437
2-- GAPDH- 16.9393409-- 16.981744-- 17.1600726 17.0270525
3-- GAPDH- 17.477777-- 17.2306059-- 17.3061118 17.3381649
4-- GAPDH- 17.3204051-- 17.0312215-- 17.1166215-- 17.1560827
1-- ACTIN-2- 15.6542578-- 16.0506853-- 15.308673-- 15.67120537
2-- ACTIN-2- 15.4098156-- 15.4334925-- 15.0512194-- 15.29817583
3-- ACTIN-2- 15.3326059-- 15.3368817-- 15.2771232-- 15.31553693
4-- ACTIN-2- 15.6690638-- 15.697236-- 15.5794577-- 15.64858583


I have another question:
Do you know how many differences between CT values of three repeats of a reference gene are acceptable?

Edited by memari, 07 November 2012 - 11:56 AM.

-----
Babak Memari

#2 Trof

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Posted 07 November 2012 - 02:51 PM

Use geNorm or NormFinder to find most stable genes ( I would, but geNorm is currently not working in my 64bit Office)
But from my point of view you don't have much to choose from. 18S has the biggest problem of being too abundant, so maybe limiting the primers would help a bit, and then you only have GAPDH and actin, which is know to be problematic, you have even twice.

Ct variation between replicates is not telling you anything about the housekeeping gene's stability (if I understand correctly that those are technical replicates, not biological).

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


#3 Afiqah Alyaa

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Posted 15 November 2012 - 05:15 AM

A technician specialist told me that ideally should only vary 1-2 Ct.

#4 Trof

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Posted 15 November 2012 - 06:06 AM

Ct's of different samples using same housekeepings should fall between 1-2 cycles, not the Ct values within technical replicate of one sample.

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






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