Gelred nucleic acid gel stain

The assayed compounds were dissolved in DMSO. The
PCR master mixture consisted of 40 mM Tris-acetate pH 8.3, 25 mM MgCl₂, 4 U of Taq DNA polymerase (Sigma-Aldrich),
20 μM each oligonucleotide primer, and 2.5 mM each deoxynucleotide
triphosphate (dNTP). Inhibition studies were carried out with varying
compound concentrations. For inhibition control, ddATP at a 200 μM
concentration was used. All PCRs were done in 20 μL of reaction
volumes. To carry out the PCR assays, the constitutive gene of Yersinia enterocolitica 16S rDNA was amplified using
specific primers.
Thermocycling conditions consisted of 35 cycles
of denaturation at 95 °C for 1 min, followed by primer annealing
at 56 °C and primer extension at 72 °C for 90 seg. After
completion of the reaction, 4 μL of loading buffer 10×
were added. The amplified DNA sequences were electrophoresed for 60
min in 1% agarose gel in buffer TBE 1× (Tris-boric-EDTA, pH 8)
at 80–85 V using TBE running buffer 1×. Finally, gels
were stained using GelRed Nucleic Acid Gel Stain (Sigma-Aldrich).
Amplified DNA bands were detected visually with a UV transilluminator.
Each assay was replicated between four times.

Total RNA was extracted
using Trizol (Invitrogen, Waltham, MA) according to the manufacturer’s
instructions. The purity and concentration of the samples were checked
measuring the absorbance at 260 and 280 nm using a NanoQuant microplate
reader (BioTek, Epoch, Vermont). Only RNA samples with an Abs260/Abs280
ratio between 1·8 and 2·0 were used for gene expression
analyses. Retrotranscription was carried out with M-MLV Reverse Transcriptase
virus enzyme 200 U μL^–1 (Sigma-Aldrich) according
to the manufacturer’s instructions. Two micrograms of isolated
RNA, previously suspended in diethylpyrocarbonate-treated water, was
used. The primer design was done using PubMed database and OligoCalc
software. The gene expression levels were normalized to the levels
of the 16S rRNA housekeeping gene utilizing ImageJ 1.51n software
for relative quantification.^{35 (link)}After
completion of the reaction, 4 μL of loading buffer 10×
was added. The amplified DNA sequences were electrophoresed for 60
min in 1% agarose gel in buffer TBE 1× (Tris-boric-EDTA, pH 8)
at 80–85 V using TBE running buffer 1×. Finally, gels
were stained using GelRed Nucleic Acid Gel Stain (Sigma-Aldrich).
For inhibition control, ddATP at 200 μM concentration was used.
Amplified DNA bands were detected visually with a UV transilluminator.
Each assay was replicated between four times.

The Poly(A) tail length was determined using the Poly(A) tail length assay kit (Thermo Fisher, #764551KT) following the manufacturer's protocol. The total RNA of HEK293 NC and KO cells was extracted with TRIzol reagent (Thermo Fisher, #15596026), treated with DNase I (Thermo Fisher, #AM2222), and purified with phenol-chloroform again. 1 mg total RNA sample was added with poly(G/I) tails, reverse transcribed the poly(G/I) tailed RNA and the poly(G/I) tailed cDNA was PCR amplified. The size of PCR products can be assessed by running one-half of each PCR reaction (12.5 μl) per lane on a 5% non-denaturing polyacrylamide TBE gel. Stain gels with 50 ml GelRed Nucleic Acid Gel Stain (Sigma Aldrich, #SCT123), diluted in 0.1 M NaCl, for 1 h, and images were captured by a GelDoc Gel Documentation System (Bio-Rad, USA).