Strategies and Best Practice in Cloning Small RNAs
The discovery of the first miRNA lin-4 about 30 years ago by Victor Ambros lab may seem unimportant and irrelevant to human health since it is not conserved . However, it inevitably opened a new era in small RNA-mediated gene regulation, antivirus and gene-editing. It once again proves that any biological techniques humans developed may have been utilized by cells for millions of years. In this case, scientists used the base pairing rule to develop hybridization methods for detecting nucleic acids before realizing that cells use the same strategy to screen target nuclei acids. Apparently cells outsmart humans since cells need to balance between detection sensitivity and specificity by using small RNAs while humans are more concerned with sensitivity by using long RNA probes.
Among them, micro(mi)RNAs and small interfering RNAs (siRNA) are processed from double-stranded (ds)RNAs in RNA interference (RNAi)-related processes, which regulate host and viral genes transcriptionally and post transcriptionally; Piwi-interacting RNAs (piRNA) are processed from single stranded (ss)RNA precursors and play critical roles in maintaining genome stability in germline cells.
Unlike intact or fragmented mRNAs, small RNAs often contain distinct modifications usually at their 5' and 3' ends, which may block RNA ligation for constructing small RNA libraries. For example, animal miRNAs usually contain a monophosphate (p) group at the 5' end (5'p-RNA) and 3' hydroxyl (OH) group at the 3' end resulted from Dicer-mediated processing; plant miRNA and siRNAs are mostly 2'-O-methylated at the 3' end for protection; animal piRNAs are also 2'-O-methylated at the 3' end for protection.
RNA Quality And Quantity
Small RNA cloning can start with total RNAs or purified small RNAs. Many old protocols used purified small RNAs which can be obtained via denaturing polyacrylamide gel (PAGE) purification (6-8 M urea) or affinity-based purification The former can even reach single-nt resolution, generating small RNA fractions of better quality; the latter usually first utilizes affinity columns to remove big RNA and then column or alcohol precipitation to concentrate small RNAs of less than 200 nts In both cases, a denaturing condition containing urea or guanidine is preferred for separating small RNAs from their targets.
PAGE-based purification is still desirable for samples containing lots of degraded RNAs, which may overwhelm authentic small RNAs. For example, immunoprecipitated RNA samples may contain lots of degraded tRNAs, rRNAs, and mRNAs, and at least a small fraction of them are ligatable, likely generating a huge background noise. PAGE purification can remove this noise by selecting small RNAs of desired sizes.
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