Hipura total rna miniprep purification kit

HT1080 and HT1080/DOX cells were seeded in six-well tissue culture plate (Tarsons; 980010) at 2 × 10⁵ cells/well. After 24 h of incubation, cells were trypsinized and washed with 1X PBS twice. Total RNAs were isolated from both the cell lines using HiPurA^TM Total RNA Miniprep Purification Kit (Himedia; MB602) according to the manufacturer’s instructions. The quality and quantity of RNA were assessed as described previously in the section “small RNA isolation and qRT-PCR analysis”. The cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific; K1622). The expression of predicted target genes was quantified by performing qRT-PCR using Hi-SYBr Master Mix (Himedia; MBT074) in QuantStudio 5 real-time PCR (Applied Biosystem, Foster City, CA, USA; A34322).
The expression of target genes was also measured by qRT-PCR after transfection of HT1080/DOX cells with 20 nM piR-39980 mimic/inhibitor and HT1080 cells with 20 nM piR-39980 mimic/inhibitor. RPL13 was used as an endogenous control for normalization, and data are expressed as 2^−ΔΔCt. Primers were synthesized from IDT. Primers are listed in Supplementary Table 2.

Total RNA was extracted using HiPurA^Tm Total RNA Miniprep purification kit from Himedia laboratories. Subsequently, 2 µg of total RNA was converted into cDNA using Reverse Transcriptase and manufacturer’s instructions were followed. For QPCR (AB 7500, Step One), 2 µl of the RT product was used with gene specific primers in conjunction with SYBR green. Human18s rRNA was used as internal control and relative gene expression was calculated using 2^−ΔΔCTmethod. List of primers used in this study is tabulated in Supplementary Table 2.

Carvacrol (CAR), DAPI (4, 6-diamidino-2-phenylindole), propidium iodide (PI), and 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, United States). Caspase-9, -8 and -3 colorimetric assay kit with catalogue numbers K119, K113–25 and K106-100 were procured from BioVision, United States. Acridine orange, Ethidium bromide, RPMI-1640, fetal bovine serum (FBS), 1% antibiotic-antimycotic solution, RNase A, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and HiPurATM Total RNA Miniprep Purification Kit were purchased from Himedia India, Ltd., Mumbai, India. JC-1 mitochondrial membrane potential (MMP) assay kit was purchased from G-Biosciences, United States. All the primer sequences utilized during the study were procured from IDT, United States. FITC Annexin V Apoptosis Detection Kit was procured from BD Bioscience, PharMingen (San Diego, United States of America). DyNAmoColorFlash SYBR Green qPCR Kit and Verso cDNA synthesis kit were obtained from Thermo-Scientific, United States.

The RNA extraction of treated and untreated cells was performed using the HiPurATM Total RNA Miniprep Purification Kit (Himedia, Mumbai, India). The cDNA synthesis was performed using Verso cDNA synthesis kit (ThermoFisher Scientific, Waltham, MA, USA) and the mRNA expression level of target genes was estimatedby using DyNAmoColorFlash SYBR Green qPCR Kit (ThermoFisher Scientific, Waltham, MA, USA).The primer sequences target for this study were as follows:
Bax 5′-AAGAAGCTGAGCGAGTGT-3′   5′-GGAGGAAGTCCAATGTC-3′ (Accession number: NG_012191); Bcl2 5′-TCCATGTCTTTGGACAACCA; 5′-CTCCACCAGTGTTCCCATCT-3′ (Accession number: NM_000633.2); CyclinD1   5′-TGTGTGCAGAAGGAGGTCC-3′   5′-CCTTCATCTTAGAGGCCACG-3′ (Accession number: NM_012142.4); CDK4   5′-AGTGTACAAGGCCCGTGATC-3′   5′-ACGAACTGTGCTGATGGGAAG-3′ (Accession number: NM_000075.3); Notch-1   5’-GCAGTTGTGCTCCTGAAGAA-3’   5’-CGGGCGGCCAGAAAC-3’ (Accession number: NM_017617); Hes-1 Forward Primer: 5′-ATTCTGGAAATGACAGTGAAGCAC-3′   Reverse Primer: 5′-CACCTCGGTATTAACGCCCTC-3′ (Accession number: NM_005524.3); and GAPDH   Forward Primer: 5’-GAAGGTCGGAGTCAACGGAT TTG GT-3’   Reverse Primer: 5’-CATGTGGGCCATGAGGTCCACCAC-3’ (Accession number: NM_002046.5). GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used as an internal control and the data was evaluated by the comparative 2^−ΔΔCt threshold cycle.

Cell culture reagents, including Ham’s F-12K media, fetal bovine serum (FBS), antibiotic–antimycotic cocktail, MTT stain, Hoechst 33342, and DCFH-DA dye, were procured from Sigma (St. Louis, MO, United States). RNase-A and the HiPurA TM Total RNA Miniprep Purification Kit used were from HiMedia, Mumbai, India. The DyNAmo ColorFlash SYBR Green Quantitative Polymerase Chain Reaction (qPCR) Kit was from Thermo Fisher Scientific, Waltham, MA, United States. The primers in the study were procured from IDT, Coralville, IA, United States.