tissue: Tarsal conjunctiva gender: F disease status: control
Extracted molecule
total RNA
Extraction protocol
Qiagen All-prep kits with miRNA addition
Label
FAM
Label protocol
FAM
Hybridization protocol
n/a
Scan protocol
n/a
Description
F
Data processing
QRT PCR results processed in ABI SDS 2.4 RQ manager. Plates processed individually to generate non-normalized data using the following settings: Fluorescence off scale = ON, A well has missing data = ON, Laser power low during run = ON, Large mean squared error = >2000, Percentage of plate wells not amplified = >80, A well is empty = OFF, Bad passive reference signal > 0.6, No amplification of well = OFF, High noise spikes = >1, High relative noise = >4. Set manual threshold to 0.05 & automatic baseline --> ANALYSE ALL. Data exported and processed in R using bioconductor HTqPCR package. Samples with a global miRNA median cycle threshold of 40 and samples with poor correlation within phenotype groups were excluded. A and B card data were processed seperately due to a different number of samples in each group as a result of filtering. Data were normalised with nr.norm (rank invariant normalization) as it gave the lowest coefficient of variation for all data and the highest sample distribution homology across all of the normaliation procedures tested. Data were tested for differential expression with Limma.
