miRNA-Seq

For microRNA profiling, the GSL currently uses the Illumina platform. Samples can be indexed and sequenced many per lane.

Sample Submission Requirements

The method of total RNA isolation is important to make sure the microRNA fraction has been retained. Trizol isolation alone does retain the microRNA, but if followed by an RNAeasy clean-up column, the microRNA fraction will be lost. There are many commercial kits available for RNA isolation; for example, Qiagen has a kit directed specifically at microRNA retention.

Submission requirements are 1-10ug of total RNA at a concentration of 250ng/ul to 1ug/ul in 10mM Tris or Qiagen EB. A large amount of RNA is requested since a microRNA enrichment/purification on the total RNA is the first step of the microRNA-Seq protocol. The acceptable range of input is broad because certain tissues have high amounts of microRNA while others have little. Smaller RNA inputs may be possible to work with but the amount of microRNA present in the samples would need to be verified first by bioanalysis. The protocol allows up to 6ul of input RNA volume, so the sample may not be too dilute. If so, a fee of $25/sample will be charged to concentrate the sample.

All RNA samples will undergo quantitation and bioanalysis to confirm quality. RNA should have a RIN of 6.0 or greater, so that degradation products don't contaminate the microRNA fraction.

GSL miRNA-Seq Data

All demultiplexing (i.e. the sorting of indexed reads) is included in the cost of basic RNA-Seq, with users receiving .fastq files for each sample. miRNA samples are run under single-end, 50bp conditions. Additional fees are charged for analysis, including alignment.