Small RNA sequencing is a protocol developed to enable the discovery and profiling of microRNAs (miRNAs) and other non-coding RNAs, which unlike with microarrays or qPCR, doesn’t require a priori knowledge of sequence. The protocol is targeted toward short inserts because microRNAs are in the 18-23 bp range and piRNAs, snoRNAs, tRNAs are all under 100bps. The method takes advantage of the fact that microRNA and other small non-coding RNAs have a 5’phosphate and 3’hydroxyl group in their native state. This allows the use of RNA ligase to add on single stranded adapters that act as reverse transcription primer and PCR binding sites. Caution should be observed as sequencing data containing differential expression of small RNAs often does not correlate well with qPCR or microarray analysis, even when the same sample is used. This has been attributed to the bias RNA ligase has for particular adapter sequences. Methods to ameliorate these issues are being developed.
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See Genohub's up-to-date list of available Small RNA library prep services
Bioo Scientific’s Illumina compatible NEXTflex Small RNA Seq v2 Kit is currently the only kit on the market that has a solution for reducing ligation associated bias. The kit utilizes 3’ and 5’ adapters with 4nt random ends which gives T4RNA Ligase a full complement of adapter combinations during the ligation step. This reduces the chance for preferred adapter:microRNA ligation events, providing a more even coverage of individual small RNA species. The kit comes with all the necessary reagents to transform total or isolated small RNA into libraries for sequencing. Adapter self ligation, also known as adapter dimers are reduced using an ‘Adapter Depletion Solution’ immediately after the 3’ligation and reverse transcription steps. The kit also comes with a positive control, consisting of a unique synthetic microRNA oligonucleotide. Small RNA sequencing is challenging and fraught with bias. This is the first commercial kit to address a well known issue.
The ScriptMiner kit is designed to prepare small RNA-Seq libraries from microRNA, plant miRNA and from small 5’capped and small 5’-triphosphorylated RNAs. The preparation of 1 library takes approximately 1 day. The user can start with 1-5 µg of total RNA or 100 pg of size selected small RNA. The kit is compatible with up to 12 PCR based barcodes that are available separately.
The TruSeq Small RNA kit is designed for generating small RNA libraries from as low as 1 µg of Total RNA. The kit takes advantage of the hallmark 3’ hydroxyl group microRNAs have that result from the enzymatic cleavage by Dicer (and other RNA processing enzymes) and an adenylated 3’ adapter that specifically ligates to this end. By targeting the 3’ end of small RNAs, unwanted ligation to other transcripts is reduced allowing for specific sequencing of microRNAs and other 3’OH transcript species. 12 barcoded PCR primers are included with this kit, with up to 48 unique barcodes available.
One of the key steps in all Small RNA library preparation procedures is the reduction of adapter dimers between the 3’ and 5’ adapters. While the 3’ and 5’ adapters are designed to ligate to small RNAs, self ligation can occur, resulting in preferential amplification of this smaller sized product and downstream sequencing contamination. Adapter dimers result in fewer sequencing reads of small RNAs and can significantly skew small RNA profiles. Illumina’s TruSeq Small RNA kit utilizes a hairpin oligonucleotide that has two complementary regions that hybridize to the 3’adapter, after the 3’ligation step to reduce the availability of free 3’adapter and hence reduce 3’ and 5’ adapter formation. Details can be found in their patent application.
The NEBNext Small RNA kit is designed for generating small RNA libraries from as low as 1 µg of Total RNA. The kit takes advantage of the hallmark 3’ hydroxyl group microRNAs have that result from the enzymatic cleavage by Dicer (and other RNA processing enzymes) and an adenylated 3’ adapter that specifically ligates to this end. By targeting the 3’ end of small RNAs, unwanted ligation to other transcripts is reduced allowing for specific sequencing of microRNAs and other 3’OH transcript species. The kit does not include multiplexing adapters or PCR primers.
The TailorMix kit from Seqmatic can use as little as 10 ng of total RNA to generate microRNA libraries compatible with Illumina sequencing. Using ready to use enzyme formulations, libraries can be constructed in a single day.