Supplementary Materials and methods

Polyadenylation and reverse transcription of the extracted total RNA

In brief, 2 μg of total RNA were polyadenylated using recombinant E.coli poly(A) polymerase (New England Biolabs Ltd., Ontario, Canada) in the presence of ATP (80 μmol/L) at 37 oC for 60 min, followed by a 10-min incubation at 65 oC for enzyme deactivation. Polyadenylated RNA was mixed with an oligo-dT adapter primer sequence (5’-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3’, where V=G, A, C and N=G, A, T, C) that served as primer, heated at 65 oC for 5 min to denature any secondary structures, and quickly chilled on ice. The reverse transcription reaction was performed at 37 oC for 60 min, followed by a 15-min incubation at 70 oC for enzyme deactivation. The reaction mixture contained 2 μg of polyadenylated total RNA diluted in DNase/RNase-free water, 0.25 mM oligo-dT–adapter sequence, dNTP mix (0.5 mM each), 4 μL of 5X reaction buffer [250mM Tris-HCl (pH 8.3 at 25 oC), 375mMKCl, 15mMMgCl2], 0.01M DTT, 20 U of RNaseOUT™ (Life Technologies Ltd., Carlsbad, CA, USA) RNase inhibitor, and 100 U of MMLV Reverse Transcriptase (Life Technologies Ltd.).

RT-qPCR

The sequence of the miR-15a-5p forward primer was 5'-TAGCAGCACATAATGGTTTGT-3', that of the SNORD43 forward primer was 5'-ACTTATTGACGGGCGGACA-3', and that of the SNORD48 forward primer was 5'-TGATGATGACCCCAGGTAACTCT-3'. The sequence of the common reverse primer – complementary to the oligo-dT adapter – was 5'-GCGAGCACAGAATTAATACGAC-3'. The PCR amplicons for miR-15a-5p, SNORD43, and SNORD48 were 66-bp, 93-bp, and 105-bp long, respectively. The reaction mixture contained 1 μL of 10-fold diluted cDNA, 5 μL KAPA™ SYBR® FAST qPCR master mix (2X) (Kapa Biosystems Inc., Woburn, MA, USA), and 2 μL of primers (final concentration: 300 nM each), in a final reaction volume of 10 μL. The following cycling conditions were used: a denaturation step at 95 oC for 3 min, followed by 40 cycles of 95 oC for 3 sec, for denaturation of the PCR products, and 60 oC for 30 sec, for primer annealing and extension as well as for detection of fluorescence. Finally, melting curves of the PCR products were generated by heating the reaction mixtures from 60 oC to 95 oC with a heating rate of 0.3 oC/sec and continuously acquiring fluorescence emission data, so as to distinguish the specific PCR products from primer-dimers or other non-specific products, which are characterized by a different Tm than those of the miR-15a-5p, SNORD43, and SNORD48 amplicons.

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