U6 rna

RT-qPCR was carried out using iScript ™ One-Step RT-PCR Kit with SYBR® Green (Bio-Rad Laboratories, CA) on Rotor-Gene Q real-time PCR cycler. The primers (Life Technologies) used for the target genes: Bax gene, CASP-3 gene, TP53INP1 gene and the housekeeping gene β-actin are listed in S1. Data from real-time PCR were used to calculate the relative expression of tested genes mRNA. For the normalization of all values; the β-actin gene was utilized. Finally, values were reported as fold change using Eq. (4):³¹ $$2^{\mathrm{\Delta }\mathrm{\Delta }\text{CT}}.$$
For miRNA normalization, the internal control (U6 RNA) was used and the primer set for the internal control was purchased from RiboBio Co., Ltd. All samples were run in triplicate and the relative expression of miR‑221 was quantified by the same method.

The Cy3-labeled probes (red fluorescent signal) for RNA FISH of CRNDE, 18S RNA and U6 RNA were synthesized by RiboBio (Guangzhou, China). RNA FISH was performed using fluorescent in situ hybridization kit (RiboBio) according to the manufacturer’s instructions. DAPI was used for counterstaining of nuclei (blue fluorescent signal). The cells were photographed under a fluorescence microscope (OlympusIX81).