miRNA or microRNA/Small RNA Sequencing Services
Micro RNA sequencing differs from mRNA and total RNA sequencing because it enriches for small RNA, 22 nucleotides and 30 nucleotides in length, independent of organism. These small RNA sequences are responsible for influencing protein expression and regulating transcriptional and post-transcriptional gene expression. RNA profiling will allow researchers to find novel small RNAs and transcript isoforms, characterize mutations such as coding SNPs and analyze expression of all small RNAs in each sample.
The TruSeq Small RNA sample preparation kit primarily targets microRNAs and other small RNAs that have a 5’-phosphate and a 3’-hydroxyl group, to generate cDNA from total RNA or purified small RNA. An RNA 3’ adapter modified to target microRNAs is ligated to the 3’ hydroxyl group, while an RNA 5’ adapter is ligated to the 5’ phosphate of each fragment. A reverse transcription reaction is used to create single stranded cDNA. The cDNA is then PCR amplified using a common primer and a primer containing one of 48 unique adapters, which can be multiplexed together in a single flow cell lane. Lastly, the fragments are gel purified and pooled (optional).
Please provide 1ug of high quality total RNA prepared using an RNA extraction method that retains the small RNA species. Alternatively, submit the entire fraction of small RNA purified from 1-10 ug of total RNA.
|GARP Recommended Minimum Total RNA Input||GARP Recommended Minimum Total RNA Concentration|
|1 μg||200 ng/μl|
* DNase treatment of RNA is required to avoid false positives in downstream applications.*