Rna extraction kit

The total RNA from each TNBC patient sample was extracted from 10 µm sections of formalin-fixed paraffin-embedded blocks using an RNA extraction kit (Ambion, Austin, USA) as per the manufacturer's protocols. For quality control, RNA purity and integrity were evaluated based on the absorbance ratios at 260/280 nm and 260/230 nm that were analyzed using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).
For miRNA expression analysis, 10 ng total RNA was used along with miR-34a-specific primers supplied with the miR-34a TaqMan miRNAAssay (Applied Biosystems, Foster City, USA). The complementary DNA was synthesized using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems), and qRT-PCR analysis was performed on the LightCycler 96 system (Roche, Basel, Switzerland). The U6 small nuclear 6B (RNU6B) miRNA was used as an endogenous control. Each miRNA assay was performed in triplicate. The expression of miRNA has been reported as the delta Ct value 2^−∆Ct (−∆Ct: Ct value of RNU6B – Ct value of miR-34a). The cutoff value of miR-34a was set at 0.90, which was the median expression value. We classified the tumors into miR-34a high or low based on the cutoff expression value of miR-34a.

Total RNA was extracted from liver tissue using an RNA extraction kit (Ambion; Applied Biosystems, Foster City, CA). Complementary DNA was generated by reverse transcriptase (Invitrogen, Carlsbad, CA) with 1 μg of RNA. Real-time PCR was performed in ABI 7500 PCR system using the TaqMan primer and probe sets, based on 5’ nuclease chemistry using TaqMan minor groove binder (MGB) probes (Table 1). Target mRNA levels were normalized to β-actin as an endogenous control gene.

The viral RNA samples were extracted from nasal swab sample using Ambion^® RNA extraction kit (USA) as per the manufacturer’s instructions. The one-step conventional reverse transcription polymerase chain reaction (RT-PCR) was conducted by using the Ambion^® Kit (AgPath-ID^TM One-step RT- PCR kit, USA) as per the manufacturer’s instructions. A 25 μl scale of reaction mixture consisted of 2× RT-PCR buffer 13 μl, forward and reverse primers (100 pmole/μl each) 0.5 μl, 2× RT-PCR enzyme mix 1.0 μl, template RNA 5 μl, and the rest 5 μl was nuclease-free water. The primers used for amplification of N gene of PPRV were NP3 (5ʹ-TCTCGGAAATCGCCTCACAGACTG-3ʹ) and NP4 (5ʹ-CCTCCTCCTGGTCCTCCAGAATCT-3ʹ) targeting an amplicon of 351-bp, as described by Couacy-Hymann et al. [17 (link)]. The oligonucleotide primers were obtained from Integrated DNA Technologies (Japan). The thermal profile for the RT-PCR reaction was followed by the conditions mentioned by Couacy-Hymann et al. [17 (link)]. Agarose gel electrophoresis was done for the visualization of the RT-PCR products after staining with ethidium bromide (10 mg/ml) (Sigma^®, USA).