Table 3 Detection techniques of small RNAs
From: Small RNA modifications: regulatory molecules and potential applications
Method | Verified small RNA species | Specific features of the approach | References |
|---|---|---|---|
ARM-seq | tRNA, tsRNA | AlkB treatment to remove m1A, m3C and m1G modifications in tRNA | [194] |
DM-tRNA-seq | tRNA | AlkB treatment to remove m1A, m3C and m1G modifications in tRNAs; thermostable group II intron RT (TGIRT) with high processivity to generate cDNA from highly structured tRNA adds RNA-seq adaptors by template-switching without RNA ligation | [195] |
multiplex small RNA-seq library preparation method (MSR-seq) | tRNA, tsRNA and other small RNAs | Design of a biotinylated oligonucleotide used for barcode adapter ligation, immobilization, on-bead reverse transcription, second adapter ligation and PCR; AlkB treatment removes m1A and m1G modifications in tRNAs | [196] |
CPA-seq | small RNAs including tsRNA, snsRNA, snosRNA, lncsRNA, miRNA | Use of a deacylation buffer (pH = 9.0) to remove aminoacyl residues in aminoacyl-tRNA-derived 3′-tsRNAs; Cap-Clip to remove the 5′-cap and 5′-ppp from RNAs to generate 5′-P termini; T4 PNK to reduce terminus multiplicities; AlkB and AlkB(D135S) (AlkB mix) used to remove methylation in m1A, m3C and m1G; TGIRT-III, a highly processive reverse transcriptase, used to increase the detection of sRNAs derived from tRNAs containing m1A, m3C and m1G sites | [197] |
PANDORA-seq | miRNA, tsRNA and rsRNA | AlkB treatment to remove m1A, m3C, m1G and m22G modifications in tsRNAs;T4PNK treatment to convert 5′-OH at the 5′end into 5′-P and 3′-P and 2′,3′- cP at the 3′end into 3′-OH | [198] |
AQRNA-seq | all types(tRNA and miRNA, mRNA, rRNA, etc.) | AlkB treatment to remove m1A, m1G and m1I modifications; Shrimp alkaline phosphate treatment to convert 5′-P into 5′-OH and 3′-P into 3′-OH; Adaptor ligation at the 3′end of RNAs, to resolve the issue of 5′terminal modification | [199] |
cP-RNA-seq | 5′-tRNA halves; cP-containing RNA repertoires in various transcriptomes | Gel-purified RNAs specific sizes are purified and treated with a phosphatase (CIP), followed by treatment with a periodate (NaIO4) to disrupt 3′-ends of RNAs containing 3′-P and 3′-OH ends; T4PNK to selectively capture RNAs with 2′,3′-cP at their 3′ termini | [200] |
5´XP sRNA-seq | miRNA, piRNA, tsRNA and rsRNA | Simultaneous capture of 5′-P and non-5′-P RNAs with the 5′-P RNA tagged with a barcode sequence resolved during bioinformatic analyses | [201] |