[R-sig-ME] Mixed Models - remedy for pseudoreplication?

ONKELINX, Thierry Thierry.ONKELINX at inbo.be
Tue Jul 30 15:41:33 CEST 2013

Dear Jens,

You will need to provide more details on the design of the experiment.

- do you create 6 plates on day 0 and measure them on day 1, 2, ... or do you fill a new plate each day with the content of the flasks?
- how are the flasks distributed over the plates? Each well come for a different flask? Or all wells from the same column come from one flash?

We need all the details of the design to suggest a sensible model.

Best regards,


ir. Thierry Onkelinx
Instituut voor natuur- en bosonderzoek / Research Institute for Nature and Forest
team Biometrie & Kwaliteitszorg / team Biometrics & Quality Assurance
Kliniekstraat 25
1070 Anderlecht
+ 32 2 525 02 51
+ 32 54 43 61 85
Thierry.Onkelinx op inbo.be

To call in the statistician after the experiment is done may be no more than asking him to perform a post-mortem examination: he may be able to say what the experiment died of.
~ Sir Ronald Aylmer Fisher

The plural of anecdote is not data.
~ Roger Brinner

The combination of some data and an aching desire for an answer does not ensure that a reasonable answer can be extracted from a given body of data.
~ John Tukey

-----Oorspronkelijk bericht-----
Van: r-sig-mixed-models-bounces op r-project.org [mailto:r-sig-mixed-models-bounces op r-project.org] Namens Jens Oldeland
Verzonden: dinsdag 30 juli 2013 12:21
Aan: r-sig-mixed-models op r-project.org
Onderwerp: [R-sig-ME] Mixed Models - remedy for pseudoreplication?

Dear List readers,

we are studying effects of chemical therapies on the proliferation of cancer cell lines.
My question aims at the practical application of a Mixed Effect Model (lme in R) of a particular analysis of a cell culture experiment on a 96 well plate and the problem of pseudoreplication.

A 96 well plate looks like this:
and receives treatments in the columns 3-10 with 8 wells, i.e. the replications. We repeated the experiment on 6 days, with one 96 well plate per day.
We noticed a high intra-day variability.

Since all the cells of one day on which the treatments are applied come from the same flask and the media and pharmacologic agents, with which they were treated, are coming from the same tubes, the question arises if I have to count them as pseudoreplicates.
The analysis of the data using “lme” works just fine, but I worry about the high number of degrees of freedom, which include the “pseudoreplicated” measurements.

An alternative would be to average the 8 replicates per day and use an ANOVA with an Error structure, which seems less elegant to me, since we are not including the high intra-day variability.

I would be very happy if anyone could give an adivce on how to deal with this dataset. I found different opinions in the literature, some saying that LMMs are a "remedy" for 96 well plates and some say that 96 well plates are per-se pseudoreplicated...

thanks in advance!

Dr. Jens Oldeland

Post-Doc Researcher & Lecturer @ BEE
Managing Editor - Biodiversity & Ecology

Biodiversity, Ecology and Evolution of Plants (BEE) Biocentre Klein Flottbek and Botanical Garden University of Hamburg Ohnhorststr. 18
22609 Hamburg,

Tel:    0049-(0)40-42816-407
Fax:    0049-(0)40-42816-543
Mail:   jens.oldeland op uni-hamburg.de
         Oldeland op gmx.de
Skype:  jens.oldeland

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