prevailing techniques to identify sRNAs and detect their expression. These methods are easy and still widely used in many laboratories that are modern-resource inaccessible. However, the
limitations of these approaches are the relatively lower recovery of
sRNA species and that they restricted to only small numbers of
sRNAs [6]. Next-generation sequencing (NGS) has been developed as a powerful technique to detect and quantify sRNAs [7–
10]. NGS technique has significant advantages in systematically
recovering sRNAs with high efficiency, especially the ones with
low abundance, or the ones that are expressed in specific tissue
niches, developmental stages, and physiological processes. Numerous commercial kits exemplified by the ones provided by Illumina
and NEB vendors are at hand for this purpose and wide arrays of
NGS protocols have also been customized/published. All the
methods are based on the fact that most classes of sRNAs harbor
5
0 phosphate and 3
0 hydroxyl termini that result from RNA processing. This feature distinguishes bona fide sRNAs from RNA turnover products and RNase degradation products that rather contain
a 5
0 hydroxyl and a 2
0 or 3
0 phosphate group. Thus, the bona fide
sRNAs are easily captured through ligations with a pair of oligo
adapters that provide 5
0 phosphate and 3
0 hydroxyl groups, respectively. One problem to clone sRNA library is circularization of 5
0
phosphate/3
0 hydroxyl sRNAs and even adapters themselves during the adapter ligation process. To prevent this issue, chemically
pre-adenylated 3
0 adapter deoxyoligonucleotides, which are also
blocked at their 3
0 ends through an amino group or a dideoxy
nucleotide, are widely used to avoid their circularization [7]. The
use of 5
0 -pre-adenylated adapters eliminates the need for ATP
during ligation, and thus solves the problem of adenylation of the
pool RNA 5
0 phosphate that causes circularization. Another strategy to address circularization is to use a truncated and mutant form
of T4 RNA ligase 2, Rnl2(1–249)K227Q. This T4 RNA ligase
mutant is able to prevent adenylyl transfer from the 5
0 phosphate
of 3
0 adapter to the 5
0 phosphate of the sRNA pool and subsequent
pool RNA circularization [11].
To simplify the cloning process and minimize the workload for
sRNA library construction and sequencing, essentially all commercial kits and a majority of the customized NGS methods do not
separate sRNAs from other RNA species in total RNA through a
size-fractionation process in a polyacrylamide gel. Instead, total
RNA is initially used through the adapter ligation processes and
final separation of potential sRNA-containing cDNA fragments is
conducted according to their estimated sizes in an Agarose gel.
However, the pre-adenylated 3
0 adapter provided in most of the
kits has very low concentration (i.e., 5 μM in NEBNext
® Multiplex
Small RNA Library Prep Set for Illumina
® ), likely due to the
relative technique difficulty in chemical synthesis. On the other
hand, many species of RNAs including the abundant rRNAs and
226
Di Sun et al.
limitations of these approaches are the relatively lower recovery of
sRNA species and that they restricted to only small numbers of
sRNAs [6]. Next-generation sequencing (NGS) has been developed as a powerful technique to detect and quantify sRNAs [7–
10]. NGS technique has significant advantages in systematically
recovering sRNAs with high efficiency, especially the ones with
low abundance, or the ones that are expressed in specific tissue
niches, developmental stages, and physiological processes. Numerous commercial kits exemplified by the ones provided by Illumina
and NEB vendors are at hand for this purpose and wide arrays of
NGS protocols have also been customized/published. All the
methods are based on the fact that most classes of sRNAs harbor
5
0 phosphate and 3
0 hydroxyl termini that result from RNA processing. This feature distinguishes bona fide sRNAs from RNA turnover products and RNase degradation products that rather contain
a 5
0 hydroxyl and a 2
0 or 3
0 phosphate group. Thus, the bona fide
sRNAs are easily captured through ligations with a pair of oligo
adapters that provide 5
0 phosphate and 3
0 hydroxyl groups, respectively. One problem to clone sRNA library is circularization of 5
0
phosphate/3
0 hydroxyl sRNAs and even adapters themselves during the adapter ligation process. To prevent this issue, chemically
pre-adenylated 3
0 adapter deoxyoligonucleotides, which are also
blocked at their 3
0 ends through an amino group or a dideoxy
nucleotide, are widely used to avoid their circularization [7]. The
use of 5
0 -pre-adenylated adapters eliminates the need for ATP
during ligation, and thus solves the problem of adenylation of the
pool RNA 5
0 phosphate that causes circularization. Another strategy to address circularization is to use a truncated and mutant form
of T4 RNA ligase 2, Rnl2(1–249)K227Q. This T4 RNA ligase
mutant is able to prevent adenylyl transfer from the 5
0 phosphate
of 3
0 adapter to the 5
0 phosphate of the sRNA pool and subsequent
pool RNA circularization [11].
To simplify the cloning process and minimize the workload for
sRNA library construction and sequencing, essentially all commercial kits and a majority of the customized NGS methods do not
separate sRNAs from other RNA species in total RNA through a
size-fractionation process in a polyacrylamide gel. Instead, total
RNA is initially used through the adapter ligation processes and
final separation of potential sRNA-containing cDNA fragments is
conducted according to their estimated sizes in an Agarose gel.
However, the pre-adenylated 3
0 adapter provided in most of the
kits has very low concentration (i.e., 5 μM in NEBNext
® Multiplex
Small RNA Library Prep Set for Illumina
® ), likely due to the
relative technique difficulty in chemical synthesis. On the other
hand, many species of RNAs including the abundant rRNAs and
226
Di Sun et al.