Download Improving Your Experiment Through Replication

TECHNICAL NOTE
Improving Your Experiment Through Replication
Why should I replicate?
Replication for Power and Precision
Statistical power enables you to find real differences between experimental groups. With adequate
replication, “real” differences in levels of gene expression can be distinguished from differences
caused by random variation. Without replication, it is difficult to know whether observed differences
are real or random.
Statistical precision enables you to accurately characterize gene expression for a particular
experimental unit. With adequate replication, you get a more accurate “overall” picture of expression.
Without replication, you have more random variation, leading to a less accurate picture and no way to
fully characterize the uncertainty in the data.
Where does this variation come from?
Sources of Variation
Biological
Variation Between…
Strains
Animals
Tissues
Times
Processing
Variation Caused By…
Quality of the Experimental Sample
Labeling Effects
Hybridization Effects
Background Effects
How many replicates do I need?
Replication Requirements
There is no simple guidance on the number of replicates needed. EA recommends a minimum of four
or five replications for each experimental condition and/or time point. However, more may be needed
to achieve the goals of many experiments. Some general guidance follows.
More replication is needed for…
Finding small differences in genes expressed
at modest levels
Experiments using tissue samples
Experiments with no confirmatory testing
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Less replication is needed for…
Finding gross patterns among highly expressed
genes
Experiments using cell line samples
Experiments incorporating confirmatory testing
such as Northern blots or Real-Time PCR
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Durham, NC 27713
Phone: 919-405-2248
What about pooling RNA?
Pooled RNA
A common practice involves pooling RNA from several experimental units (e.g. animals) in an effort to
achieve more representative results. While pooling RNA in a replicated experiment may indeed
improve statistical power and precision due to less variation across pooled samples, pooling RNA is
not a substitute for replicating an experiment. Consider:
•
The practice of pooling RNA does not in itself provide a way to characterize random variation
in the experiment, so replication is still needed to distinguish “real” differences.
•
Pooling RNA can distort results if, for example, a particular experimental unit is problematic
and contributes a misleading expression pattern that skews the results.
•
Pooling RNA precludes the investigator from observing potentially interesting patterns in
behavior across different animals or other experimental units.
Isn’t there a standard rule?
No Easy Answers
Standard statistical methods support sample size calculations to determine how many samples are
needed to detect a specified difference between groups with a required level of power. In concept,
this can be done for microarray experiments too. However, sample size calculations are based on a
known level of variation between samples. For microarrays, the reality is that:
(a) The expected level of variation is usually not well known in advance. Due to the high cost of
microarrays and the large number of samples needed to accurately assess variance, it is
usually not practical to follow the common statistical practice of gathering “pilot” data for the
purpose of estimating variability.
(b) Variation between samples can differ for different genes, so the ideal number of replicates
may differ as well. This makes it impossible to have a single rule that works in general,
without applying some simplifying assumptions.
What does all this mean?
Summary
Replication is an important component of any successful experiment. Because there is no standard
number of replicates that is right for everyone, in practice, cost considerations often dictate the
amount of replication that can be achieved. This document is intended to provide general guidance
for scientists considering the issue of replication. EA representatives are available to discuss
replication in greater detail for your experiment.
TN-Repl 104v1
2605 Meridian Parkway
Durham, NC 27713
Phone: 919-405-2248