The RIN: an RNA integrity number for assigning integrity values to RNA measurements
- Andreas Schroeder^{1, 3},
- Odilo Mueller^{2}Email author,
- Susanne Stocker^{1, 4},
- Ruediger Salowsky^{1},
- Michael Leiber^{1},
- Marcus Gassmann^{1},
- Samar Lightfoot^{2},
- Wolfram Menzel^{5},
- Martin Granzow^{6} and
- Thomas Ragg^{6}Email author
DOI: 10.1186/1471-2199-7-3
© Schroeder et al; licensee BioMed Central Ltd. 2006
Received: 17 August 2005
Accepted: 31 January 2006
Published: 31 January 2006
Abstract
Background
The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way.
Methods
A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability.
Results
This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN).
Conclusion
Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
Background
The RNA molecule plays a critical role in transferring information encoded in the genome (DNA) to the many different forms of proteins. After extracting RNA from cells by various methods, scientists are provided with a direct measure of cellular activity using gene expression measurement techniques. Among these, real-time PCR and DNA microarrays are the most widely used techniques.
Importance of integrity
RNA is a thermodynamically stable molecule, which is, however, rapidly digested in the presence of the nearly ubiquitous RNase enzymes. As a result, shorter fragments of RNA commonly occur in a sample, which can potentially compromise results of downstream applications [1, 2]. In order to evaluate the degree of degradation, electrophoretic methods have been applied that separate the samples according to the size of the comprised molecules.
Historically, RNA integrity is evaluated using agarose gel electrophoresis stained with ethidium bromide, which produces a certain banding pattern [3]. Typically, gel images show two bands comprising the 28S and 18S ribosomal RNA (rRNA) species and other bands where smaller RNA species are located. RNA is considered of high quality when the ratio of 28S:18S bands is about 2.0 and higher. Since this approach relies on human interpretation of gel images, it is subjective, hardly comparable from one lab to another, and the resulting data cannot be processed digitally.
Towards an automated approach
In 1999, the Agilent 2100 bioanalyzer was introduced for the separation of DNA, RNA, and protein samples. It has since become a mainstream technique for the analysis of RNA samples. The bioanalyzer is an automated bio-analytical device using microfluidics technology that provides eletrophoretic separations in an automated and reproducible manner [4]. Tiny amounts of RNA samples are separated in the channels of the microfabricated chips according to their molecular weight and subsequently detected via laser-induced fluorescence detection. The result is visualized as an electropherogram where the amount of measured fluorescence correlates with the amount of RNA of a given size.
Since data are produced in a digital format, they can be easily re-processed to allow additional calculations based on the acquired raw data. When first released, the Biosizing Software calculated the ratio of the two ribosomal bands, following the commonly used approach for RNA integrity assessment. However, although assessing RNA quality with the bioanalyzer proves to be far superior to the slab gel based approach, the usage of ribosomal ratio for RNA quality assessment has several shortcomings. In many cases, ribosomal ratios showed only weak correlation with RNA integrity [2, 5] and more in-depth analysis of the electrophoretic traces requires expert knowledge and is of a subjective nature.
Techniques for measuring RNA integrity
Integrity measures for RNA
The degradation process of RNA is only partly understood since it is dependent on the type of RNase that is present and is often combined with fragmentation processes. Also, the quality of RNA in a given experiment can vary extensively from one extraction to another and needs to be under constant surveillance. Using precise analytical instrumentation such as the Agilent 2100 bioanalyzer, human experts are capable of distinguishing RNA samples of different quality by examining electrophoretic traces and assigning integrity values or integrity categories [2]. Note that the degradation of RNA is a continuous process, which implies that there are no natural integrity categories.
In this situation, machine learning methods can be applied to data from experimental measurements to learn an implicit model of RNA integrity in order to achieve a user-independent method for assigning integrity values. That is, the algorithm extracts the relationship between characteristics of the measurement, or features, and the expert-assigned categories.
- 1.
Data labeling and preprocessing
- 2.
Extracting features from the electropherogram
- 3.
Selecting an appropriate feature combination
- 4.
Training prediction models and selecting the most promising model
Results
Sample preparation and data basis
Description of the data. The table summarizes the distribution of electropherograms over categories and the percentages for training and test sets are given.
Category | Training set | in % | Test set | in % | Total | in % |
---|---|---|---|---|---|---|
1 | 190 | 20.3% | 77 | 19.2% | 267 | 20.0% |
2 | 84 | 9.0% | 35 | 8.7% | 119 | 8.9% |
3 | 17 | 1.8% | 8 | 2,0% | 25 | 1.9% |
4 | 27 | 2.9% | 8 | 2.0% | 35 | 2.6% |
5 | 17 | 1.8% | 12 | 3.0% | 29 | 2.2% |
6 | 81 | 8.6% | 35 | 8.7% | 116 | 8.7% |
7 | 80 | 8.5% | 31 | 7.7% | 111 | 8.3% |
8 | 188 | 20.1% | 81 | 20.1% | 269 | 20.1% |
9 | 117 | 12.5% | 53 | 13.2% | 170 | 12.7% |
10 | 136 | 14.5% | 62 | 15.4% | 198 | 14.8% |
Total | 937 | 402 | 1339 |
Applying our method described below to the data basis yields a sorted list of features, which was used to construct feature spaces for training regression models. Furthermore, results are given for models based on features proposed in the literature. Finally, we show the correlation of the RIN with the outcome of real-time PCR experiments.
Feature selection
The total RNA ratio was selected as first feature covering 79% of the entropy of the categorical values. The next two features contribute information about the 28S-region: 28S peak height and the 28S area ratio.
Iterative feature ranking. The table gives the results of the iterative forward search. The 2nd column shows the percentage of information in the input vector compared to the entropy of the target. The 3rd column gives the values for single features and the 4th column the rank of the single features among all features. Feature names [see Additional file 2]: 1. total.rna.ratio 2. fr28s.height 3. fr28s.area.ratio 4. ratio1828.fast 5. reg5s.endval 6. fast.area.ratio 7. fr18s.found 8. all.mean.median
Step | MI(X,T)/H(T) (Combination) | MI(X_{ i },T)/H(T) (single feature) | Rank of single feature |
---|---|---|---|
1 | 0.79 | 0.79 | 1 |
2 | 0.79 | 0.50 | 38 |
3 | 0.81 | 0.74 | 3 |
4 | 0.83 | 0.55 | 19 |
5 | 0.83 | 0.46 | 51 |
6 | 0.85 | 0.60 | 10 |
7 | 0.85 | 0.47 | 48 |
8 | 0.86 | 0.50 | 39 |
Model training
The feature selection procedure provides in each step the local optimal additional feature, which will not necessarily lead to the globally best combination. In the later iteration steps, several candidate features provide the same gain in information about the target and there is some randomness in the final selection. Explorative searching for the best combination is intractable because of the computational costs of the combinatorial search. In an additional, manual optimization step application knowledge was used to substitute some features by plausible alternatives. Feature 3 and 4 were replaced by the area ratio in the fast region (fast.area.ratio). Additionally the marker height was selected. In the normalized electropherogram, the marker height allows to detect strongly degraded samples, because it is the only part of the signal which differs from the background noise. This combination also has a relative MI value of 0.83, but the best model with 5 hidden neurons had a log value for the evidence of -42. It reaches a cross-validation error of 0.25 and a test error of 0.25, which is slightly better compared to the results from figure 4. Both models perform equally well, the later one was chosen for the final implementation in the expert software for the sake of simplicity.
Comparison of model results. The table summarizes the results for three different models on a subset of 439 samples. The amount of information provided by the features is compared as well as the model evidences and the cross validation error. The values in parentheses give the results for a reduced sample set, which contains no samples labeled BLACK with the degradometer software.
28S/18S ratio | degradation factor | RIN model | |
---|---|---|---|
MI (X;T)/H(T) | 0.43 (0.63) | 0.88 | |
log evidence | -1457 | -577 (-121) | +3.3 |
MSE (CV) | 6.3 | 2.83 (0.60) | 0.15 |
If the samples that are labeled BLACK by the degradometer software are removed from the data set (N = 186, 42%), the relative MI value increases to 0.63, the evidence reaches a value of -121, whereas the cross validation error is at 0.60, which is still four times higher than for the RIN model.
Model evaluation
If a model is supposed to extract a relationship from experimental data, it is helpful for the model evaluation to explore the data in the most important two dimensions, as well as to check for large error values which correspond to categorical misclassifications. Furthermore, the model prediction can be cross-checked against control parameters of follow-up experiments, like RT-PCR.
Visualization of decision boundaries
The 2D-visualization of total RNA ratio and 28S peak height shows, that we can clearly separate high integrity values from low integrity values. The categories form clusters in this space. However, as mentioned in the previous section, the borderline between categories is not sharp, which is due to the fact that degradation is a continuous process.
Categorical misclassifications
Simple features like the ribosomal ratio which cover only one aspect of the degradation process tend to have larger errors for certain groups of experiments. That is, they cannot distinguish very well between the categories. It is very useful to check, that only a few experiments are interchanged over more than one categorical border, that is, the model covers all aspects of the degradation process. Misclassifications are measured by Receiver Operating Characteristics (ROC, cf. [7]) for distinguishing electropherograms from different groups of categories, whereas the value of the area under the ROC-curve (AUC: area under the curve) is a balanced measure for the classification error.
We briefly and informally describe how a ROC curve is constructed. The electropherograms are sorted into a ordered list according to the integrity measure estimated by the model. For a fixed classification task, a ROC curve is constructed as exemplarily described in the following for the task of distinguishing electropherograms of category 10 from all other categories: walk through the sorted list in descending order. In each step check if the considered item is in category 10 or not according to the original expert label. If it is true, draw a line fragment in vertical direction, if it is false draw it in horizontal direction. Perfect separation of category 10 from the others would imply that the ROC curve shoots up vertically on the y-axis to the maximal value, before the first horizontal step is taken. Random assignment of electropherograms would result in a ROC curve that corresponds to a diagonal line from the origin to the right top corner. A ROC curve gives a balanced measure of the model performance by integrating over all possible classification borders. Each border corresponds to a specific ratio of sensitivity to specifity, i.e. a specific point on the ROC curve.
Proportion of misclassified samples. The table shows an detailed analysis of sample assignment to integrity categories. Each category c_{ k }is tested against a series of set union ∪c_{ i }of all other categories i with i = 1,...,k - 1. The RIN of each electropherogram is rounded up or down to the closest categorical value. The classification performance for assigning electropherograms to the two resulting sets is then measured by Receiver Operating Characteristics, more precisely by the area under the curve (cf. fig. 5). There are always a few interchanges between adjacent categories, but rarely interchanges over two categorical borders. Values are rounded to two decimals.
9 | 8 | 7 | 6 | 5 | 4 | 3 | 2 | 1 | |
---|---|---|---|---|---|---|---|---|---|
10 | 0.96 | 0.98 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
9 | 0.96 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |
8 | 0.98 | 0.99 | 1.00 | 1.00 | 1.00 | 1.00 | 0.99 | ||
7 | 0.98 | 0.99 | 0.99 | 0.99 | 0.99 | 0.98 | |||
6 | 0.96 | 0.98 | 0.98 | 0.99 | 0.98 | ||||
5 | 0.95 | 0.97 | 0.98 | 0.98 | |||||
4 | 0.98 | 0.99 | 0.98 | ||||||
3 | 0.99 | 0.98 | |||||||
2 | 0.95 |
Correlation with the outcome of experiments
Correlation of RIN values with downstream experiments is of critical importance. On the one hand, a good correlation will demonstrate the validity of this approach. On the other hand, it allows determination of threshold values in order to get meaningful downstream data. For two-color microarray experiments, this could mean for example that the two input samples should not differ by more than a given number of RIN classes, while the lowest acceptable RIN can be determined as well.
Discussion
Because of the critical influence of RNA integrity on downstream experiments, there is a strong need for a reliable, reproducible, and standardized approach to classify the quality of RNA samples. The long time standard consisting in a 28S to 18S peak ratio of 2.0 was shown to provide only weak correlation with RNA integrity.
The Agilent 2100 bioanalyzer, a bio-analytical device based on a combination of microfluidics, microcapillary electrophoresis, and fluorescence detection, provides a platform to record the size distribution of molecules, e.g., RNA, in a digital format. Since this approach is highly reproducible and automated, it provides the basis for an automated, user-independent, and reproducible approach to evaluate the integrity of RNA samples using a software algorithm.
For the development of the RNA Integrity Number algorithm, a total of 1208 RNA samples from various sources and of different degradation states was analyzed. After assigning the samples to 10 different categories ranging from 1 (worst) to 10 (best), methods from information theory were applied to calculate features describing the curve of the electropherogram. In the following step, features were selected for further processing that showed high information content about the task to distinguish the 10 categories. These features were then taken as input variables for a model-training step. Here, using a Bayesian learning approach to select the most probable model, several models were trained utilizing artificial neural networks and the best was chosen for prediction of previously unseen test data. The result produced by this procedure is an algorithm called RNA Integrity Number (RIN).
Analysis of the RIN model
- 1.
The total RNA ratio measures the fraction of the area in the region of 18S and 28S compared to the total area under the curve and reflects the proportion of large molecules compared to smaller ones. It has large values for categories 6 to 10.
- 2.
The height of the 28S peak contributes additional information about the state of the degradation process, i.e. during degradation, the 28S band disappears faster than the 18S band. Therefore, it allows detection of a beginning degradation. It has largest values for categories 9 and 10, and zero values for categories 1 to 3.
- 3.
The fast area ratio reflects how far the degradation proceeded and has typically larger values for the categories 3 to 6.
- 4.
The marker height has large values for categories 1 and 2 and small values for all other categories since short degradation products will overlap with the lower marker.
Comparing the approaches
Using a single simple feature to judge RNA Integrity was already shown to be insufficient [2, 5]. While focusing on one aspect of the electropherogram allows for a rough orientation about the integrity, it is still subjective to a high degree. Linear models based on these features show a mean squared error that is four to sixty times higher (degradation factor resp. 28S/18S ratio) than compared to the proposed approach.
The reason for this tremendous difference lies in the fact, that neither the 28S/18S ratio nor the degradation factor reflect all properties of the RNA degradation process. For example, several samples of integrity category 10 are labeled BLACK from the degradometer software as they have low signal intensities. This happened for 42% of the samples under consideration, which are all samples that were under investigation for microarray experiments. The degradation factor contains similar information as the fast area ratio, which reflects typical characteristics of categories 3 to 7. The high ribosomal ratio is useful to detect a certain amount of high quality samples, but the categorization is not valid for all of them. Using several features which complement one another and allow for a non-linear weighting of these features allowed to reduce the error to a minimum value which is in the order of the natural noise in the target data. The noise results from using a categorical grid for a continues process as well as from a few abnormalities. Interestingly, almost no interchanges over more than one categorical border are observed. Thus, the classification errors appear almost only at the borderline between two categories, which was also difficult for humans to decide, when labeling the data.
Availability of the RIN model
The Agilent 2100 bioanalyzer system software can be downloaded from Agilent's webpage [see Additional file 1]. Version B.01.03 and later will allow for measurement reviews (free of licenses) including the calculation of the RNA integrity number [8]. Up-to-date information about the RIN-project can be found at the RIN web site [9].
Conclusion
This article investigates an automated procedure for extracting features from signal measurements, selecting suitable subsets of features and extracting a functional relationship to target labels of experiments. We demonstrated that the application of the methodology to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer provides a predictive model for RNA integrity, the RIN. The generalization error is as low as the natural noise in the target values and apparently lower than for ribosomal ratios.
Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, Furthermore, it was demonstrated that the RIN can be correlated with the outcome of downstream experiments, i.e. by performing this quality assessment step users can prevent themselves from erroneous results and loss of money and resources.
We conclude, that the RNA integrity number is an important tool in conducting valid gene expression measurement experiments as real-time PCR or DNA microarray, that is already widely and successfully used since the release of the β-version. It is largely free from instrument and concentration variability, thereby facilitating the comparison of samples between different labs. For example, the RIN can be assigned to a RNA sample before shipping from a lab and the same quality control can be performed in a user-independent way at the destination lab.
Methods
Data labeling and preprocessing
For classification of RNA integrity, ten categories were defined from 1 (totally degraded RNA) to 10 (fully intact RNA). Figure 2 illustrates qualitatively the differences between the categories. Each of the 1208 samples was assigned manually to one of the categories by experienced expert users.
The categorical values provide the target values for the adaptive learning steps. The assignment to categories was very carefully done as it is critical for the performance of the resulting algorithm. Especially for RNA samples at the borderline of two adjacent integrity categories, the assignment to each of the two categories could be justified but one had to be selected. This reflects a natural randomness which is inherent in a gradual process like RNA degradation. However, such random noise in the target values can easily be handled by the learning model, which assumes noise in the target data.
Detecting abnormalities in the electropherogram is another important preprocessing step to get a clean set of training samples. Various anomalies can disturb the usual shape of an electropherogram, e.g., ghost peaks, spikes, wavy baseline, and unexpected sample type. They were observed in approx. 5% of the samples. To separate anomalies from normal samples, several simple detectors were constructed. Each detector performs a linear classification based on a threshold value. Spikes, for example, can have a large peak height but have very narrow peak width; they appear as very sharp peaks. Normally, the largest peaks in the electrophoretic traces are located at the 18S and 28S bands but compared to a spike are significantly broader. If a high peak does not cover the minimal requested area, it is rejected as a spike and marked as abnormal. Applying these detection criteria to the data set returned 117 electropherograms as abnormal. Eleven abnormal samples could not be detected for example, because a spike arose near the 28S peak and could not be identified as such. All of them were assigned a sensible label and put in the test set. This reflects the natural occurrence of such effects in the test phase.
If a critical anomaly is detected, the RIN is not computed. Instead, an error message appears to the user. If a non-critical anomaly is detected, the RIN is computed and a warning to the user is displayed [10, 11]. Baseline correction and normalization are applied to the electropherogram prior to the actual feature extraction process. These functions are standard features of Agilent's Expert Software [12]. The baseline is a constant background signal of the electropherogram and its level may significantly differ between different electropherograms. The baseline-corrected signal is then normalized. For height related features it is normalized to the global maximum of the 5S-region to precursor-region. For area related features it is normalized to the global signal area in the 5S-region to precursor-region. The pre-region, marker-region and post-region are intentionally not considered critical elements of the electrophoretic trace since they don't contain critical information about the RNA degradation process.
Feature extraction
The aim of this step is to define and extract informative features from the electropherograms. For this purpose, each electropherogram is divided into the following nine adjacent segments covering the entire electropherogram: a pre-region, a marker-region, a 5S-region, a fast-region, an 18S-region, an inter-region, a 28S-region, a precursor-region and a post-region (Fig. 8). The subdivision is based on the peak table and the fragment table as computed by Agilent's Expert Software.
Each of these segments can then be considered separately yielding a number of local features. Several iterations of generating and evaluating features showed that it is sufficient to extract a set of specific features reflecting statistical properties, such as average and maximum heights, areas and their ratios as well as signal-to-noise ratios to cover for the information contained in the different regions of an electropherogram.
In addition, several global features were extracted, i.e. features that span several segments. Among these, the average and maximum height, areas and their ratios, total RNA ratio and the 28S area ratio are the most important features. Both have been used as criteria for RNA integrity assessment in the past. The total RNA ratio is the ratio of the area of the ribosomal bands to the total area of the electropherogram, whereas 28S area ratio measures only the fraction of 28S-fragment. This set of features extracted from the electropherograms and their manually assigned RNA integrity categories form the knowledge base for the following steps of the algorithm. Additional file 2 contains a complete description of the set of features.
Feature selection
In practice, only a limited amount of data is available for determining the parameters of a model. Therefore, the dimensionality of the feature vector must be in a sensible relation to the number of data points, i.e. electropherograms. Each additional feature increases the information content of the input about the target, but decreases the number of data points per dimension in order to determine the parameters. This means that the class of functions in which the solution is searched increases fast ('empty space phenomenon') [13]. Estimating a dependency between the features and the target will be more robust when the input-target space is low-dimensional. A rough orientation to determine the maximum number of features for training a prediction model is given in [14]. For a data set of about 1000 experiments the search is therefore restricted to vectors which combine at most eight features. To select the most promising features from our candidates, we use a forward selection procedure based on mutual information. The mutual information MI (X ; T) of two random vectors X and T is defined as the Kullback-Leibler distance between the joint distribution p(x, t) and the product p(x)p(t):
$MI(X;T)={\displaystyle \int {\displaystyle \int p(x,t)\cdot \mathrm{log}\frac{p(x,t)}{p(x)p(t)}dxdt\left(1\right)}}$
and measures the degree of stochastic dependency between the two random vectors [15]. This can be expressed in terms of entropies as
MI (X; T) = H(T) - H(T|X) (2)
which describes the reduction in the uncertainty or entropy H(T) of a target vector T due to the knowledge of X. To compute the required densities in equation 1 from the empirical data, we use non-parametric kernel estimators as described in [14, 16].
As the first step in the selection procedure, a feature f is selected from the set of candidates which has maximal mutual information with the target for the given data set. In each subsequent step, one of the remaining features is selected which maximizes the mutual information with the target when added to the already selected features. This way the features are ranked by the amount of information they add about the target.
The result of the feature selection process is a sorted list (cf. table 2 which allows constructing a nested series of feature spaces with growing dimensionality up to the maximal amount of d = 8 features:
{f_{1}} ⊂ {f_{1}, f_{2}} ⊂ {f_{1}, f_{2}, f_{3}} ⊂ ... ⊂ {f_{1},..., f_{8}}
Model training
After the nested series is constructed, regression models are trained for each feature space. Neural networks are used as semi-parametric regression models [16, 17].
The functional relationship between the input features and the target, which has to be learned from the empirical data, must generalize to previously unseen data points, i.e. electropherograms of newly measured RNA. The theory of regularization shows that approximating the target as good as possible on the training data, for example, by minimizing the mean squared error E_{ D }, is not sufficient: it is crucial to balance the training error against the model complexity [17]. Therefore, we train the neural networks to minimize the regularized error E = βED + αE_{ R }. The regularization term E_{ r }measures the model complexity, taking into account the weights w_{ k }in the network. We choose the weight-decay $\frac{1}{2}{\displaystyle \sum {w}_{{k}^{2}}}$ as the regularization term. The factors α and β are additional control parameters, i.e. hyperparameters.
We apply a Bayesian approach to determine the weights w_{ k }, and the parameters α and β during training as described elsewhere [16, 18]. Given the training data D we want to find the 'best' model M(Θ) with parameter vector Θ. This is expressed in Bayes' theorem as follows
$p(\Theta |D)=\frac{p(D|\Theta )p(\Theta )}{p(D)}\left(3\right)$
The best model maximizes the posterior probability, i.e. we want to find Θ* such that p(Θ*|D) ≥ p(Θ|D) ∀ Θ.
In case of neural networks, the parameter vector consists of the weight vector w, the hyperparameters α and β as well as the topology of the network. In general, we cannot determine all these parameters at once.
For that, the Bayesian framework proceeds in a hierarchical fashion. While keeping all other parameters fixed on the first level, we search for an optimal weight vector which maximizes the posterior probability p(w|D) using the fast optimization algorithm Rprop [19].
On the second level, optimal weighting coefficients α and β are determined which again maximize the posterior probability p(α,β|D). This is done iteratively: we fix α and β and optimize the weights w_{ k }as described above. Afterwards, we re-estimate α and β and keep alternating several times between optimizing the weights and updating α and β (see [16] for the update rules and mathematical details).
On the third level, we can compare networks with different topologies. Using Bayes' rule we can write the posterior probability of a model $\mathscr{H}$ as P($\mathscr{H}$|D) = p(D|$\mathscr{H}$)·P($\mathscr{H}$)/p(D). If we assign the same prior P($\mathscr{H}$) to every model, it is sufficient to evaluate the quantity p(D|$\mathscr{H}$), which is called the evidence for $\mathscr{H}$[17, 18]. Marginalizing over α and β and making use of the posterior probability for α and β from the second level provides an equation for the logarithm of the evidence ln p(D|$\mathscr{H}$), which depends on the training error, the possible symmetries resulting from the network topology, the size of the hyperparameters α and β, the number of samples and the number of weights, the second derivative of the error (Hessian matrix) respective its determinant, as well as the eigenvalues of the Hessian, thus reflecting the balance between these terms (cf. [16]).
To find the model which best explains the given data set i.e. with maximal evidence, we systematically vary the number h of hidden units in the networks from 0 to 8. All hidden units with tangens hyperbolicus as activation function are in a single layer, restricting the search space to models with moderate non-linearity. The best topology is selected as the topology with the highest evidence in average over a 10-fold cross-validation procedure on the training data. This topology is then used to train the final model on the whole training set. Figure 6 shows a strong correlation between the logarithm of the model evidence and the cross-validation error, pointing out that the evidence is a sensible selection criterion.
Declarations
Acknowledgements
We would like to extend special thanks to our collaboration partners Ambion Inc. and the Resource Center for Genome Research (RZPD), Germany. We would also like to acknowledge all the researchers who tested the RIN algorithm and provided valuable inputs. With special gratitude, we would like to acknowledge Christine Miller and Suad Diglisic from the Johns Hopkins Medical Institute for providing the real-time PCR data used in this study.
Authors’ Affiliations
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