Rneasy mini kit reagent

Total RNA was isolated from BC cases, mastopathy samples and cell line deposits (MCF-7, SK-BR-3, MDA-MB-231, MDA-MB-468, MCF-10A) using RNeasy Mini Kit reagents (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The NanoDrop1000 spectrophotometer (ThermoFisher, Waltham, MA, USA) was used to measure the concentration and purity of RNA. Reverse transcription reactions were performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in the SimpliAmpTM Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The conditions were as follows: 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C.

Frozen Symbiodinium sp. KB8 cells were ground to a fine powder under liquid nitrogen using a mortar and pestle. Total RNA in the frozen powder was purified with RNeasy Mini kit reagents (Qiagen, Tokyo, Japan). cDNA was synthesized with PrimeScript 1st strand cDNA Synthesis kit reagents (Takara, Shiga, Japan) and total RNA as the template. To obtain the first half of the VLKP gene, the forward primer was 5'-ATGAACGCGGCCACGGCCTTTG-3' and the reverse primer was 5'-TCAAATAACGATGGCTGTCTCTTCAGCC-3'. RT-PCR was carried out using KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) and the PrimeScript-synthesized cDNA as the template. Using In-Fusion HD Cloning kit reagents (Takara), the reverse-transcribed amplicons were inserted into a SmaI-digested pQE-32 vector downstream of an encoded, in-frame, N-terminal His₆ tag (Qiagen). Sequencing of the plasmid confirmed correct insertion of the amplicon.

Measurement of HIF-1α and VEGF messenger RNA (mRNA) expression was done using one-step RT-PCR. Samples were extracted using the RNeasy Mini Kit reagent from Qiagen. RT-PCR was performed using the Rotor-Gene Q Software 2.3.1.49 real-time PCR system from Qiagen with the SensiFAST SYBR No-ROX one-Step Kit for HIF-1α and VEGF. For mixing the material into a PCR tube, use SensiFAST SYBR (2X) 10 µL composition, Primer F 0.8 µL, Primer R 0.8 µL, Reverse Transcriptase Enzyme 0.2 µL, Ribosafe RNAse Inhibitor 0.4 µL, Nuclease Free Water 5.8 µL, and RNA Extract 2 µL. Insert the PCR tube and then set the RT-PCR cycle starting with the first incubation at 45°C for 10 minutes, followed by a second incubation at 95°C for 2 minutes, then denaturation 40 cycles at 95°C for 5 seconds, and extension at 60°C for 20 seconds. Instructions per manufacturer's protocol must be strictly followed.
The primary sequences are as follows:
The mRNA expression was calculated using the Livak 2(−ΔΔCT) formula, by comparing the Ct values of the treatment group with the control group.
21 (link)
Gene expression value ≥ 1 indicates that gene expression has increased compared with control. β-actin is used as internal control. ΔCT = Ct target gene – Ct housekeeping gene. ΔΔCT = ΔCT treatment – ΔCT control. Gene expression = 2(−ΔΔCT).

The total RNA was isolated from WDL-AuNP-treated RIN-5F cells by using RNeasy mini kit reagent (QIAGEN). The isolated RNA was quantified by Nanodrop. A total of 2 µg of RNA was used to analyze RT-PCR. Two-step RT-PCR kit was used to convert cDNA from mRNA template by Oligo(dT), deoxyribonucleotide triphosphate (dNTPs), and reverse transcriptase. All the components were mixed with reverse-transcriptase buffer along with DNA primer for an hour at 37 °C. Once the cDNA conversion was over, the standard PCR was run by gene-specific oligonucleotide primers for IR (224 bp) forward 5′-GCC ATC CCG AAA GCG AAG ATC-3′ reverse 5′-TCT GGG TCC TGA TTG CAT-3′(Pubmed accession number: NM_017071), IRS-1(336 bp) forward 5-GCC AAT CTT CAT CCA GTT GCT-3′ reverse 5′-CAT CGT GAA GAA GGC ATA GGG-3′, GLUT-2 (238 bp) forward 5′-CTC GGG CCT TAG GTG TTC TTC CTT-3′ reverse 5′-TGG TTC CCT TCT GGT CTG TTC CTG-3′ (Pubmed accession number: NM_012879) and β-actin (96 bp) forward 5′-AAG TCC CTC ACC CTC CCA AAA-3′, reverse 5′-AAG CAA TGG TGT CAC CTT CCC-3′ (Pubmed Accession number: V01217;J00691) followed by the initial PCR activation at 95 °C for 5 min. Gel electrophoresis was carried out to measure expression of genes by densitometric scanning. The band intensity of each gene was normalized against β-actin by densitometer (Bio-Rad Lab Inc., Hercules, CA, USA).