Nanodrop nd 100 spectrophotometer

In total, 2.5 mL of umbilical cord blood and 2.5 mL of maternal peripheral blood were collected at the time of delivery into PAXgene whole blood RNA tubes (PreAnalytix) and stored at 25 °C for at least 2 h, at − 20 °C for 24 h, and at − 80 °C until processing. The total RNA was extracted using a RNeasy Protect Animal Blood Kit (Qiagen) according to the manufacturer’s instructions. The RNA concentration and purity were measured using a NanoDrop ND100 spectrophotometer (Thermo Scientific) and BioAnalyzer 2100 system (Agilent).

Pellets from VCaP-spiked female urine and VCaP-spiked LNCaP cell mixture were resuspended in 100 μL of extraction buffer. RNA was isolated using a PicoPure RNA isolation kit (Life Technologies), according to manufacturer’s protocols. RNA quality was measured using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) and a NanoDrop ND-100 spectrophotometer (Thermo Scientific).

Total RNA was isolated from pelleted blood cells (20 µL), spleen and kidney samples (25 mg) by using QIAzol Lysis Reagent (Qiagen, Hilden, Germany), TissueLyser II (Qiagen) with 5 mm steel beads for 2 × 5 min at 25 Hz followed by chloroform addition and collection of the aqueous phase. RNeasy QIAcube Kit (Qiagen) was used for automated RNA isolation of the aqueous phase as described by manufacturer. RNA was quantified in a NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cell free samples (plasma), 10 µL plasma was diluted in 130 µL PBS and QIAamp Viral RNA Mini QIAcube Kit (Qiagen) used according to the manufacturer instructions. Isolated RNA was eluted in 60 µL RNase-free water and stored at −80 °C until further use.

Venous blood was collected in 0.5 M EDTA vial and stored at −80°C. Genomic DNA extraction for molecular genetic studies was performed using the commercially available extraction kit (Bangalore Genei, India) and was stored at −80°C. DNA concentration was measured with a Nanodrop ND-100 Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE).

To study the differential gene expression, irradiated (2 Gy) and sham-irradiated cell cultures of the RS and the RR cell line were incubated at 37 °C for 4 and 24 h and 14 days. After that, total RNA extraction was carried out with the SPLIT RNA Extraction kit (Lexogen Gmbh, Vienna, Austria) according to the manufacturer’s instructions. RNA concentration and purity for each sample were verified with a Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The ratios of absorbance 260/280 nm and 260/230 nm were ∼2 for all the samples, and RNA concentration was ∼200 ng/μL per sample. RNA integrity was determined with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA Integrity Number (RIN) values close to 10 were obtained, indicative of high quality RNA samples. Pure RNA samples were stored at −80 °C until cDNA libraries were prepared.

For the gene expression analysis, 2 × 10⁵ freshly sorted Gdeg^high and Gdeg^low KLM1 cells were used. Total RNA was extracted from each cell type with QIAZOL, an RNeasy Micro Kit, and QIAshredder spin columns (QIAGEN, Hilden, Germany). The integrity of the RNA obtained was assessed with a NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA), a 2100Bioanalyzer, and an RNA6000Pico Kit (Agilent Technologies, Palo Alto, CA, USA). According to the GeneChip®3’IVT Express Kit, P/N702646 Rev.7 protocol (Affymetrix, Santa Clara, CA, USA), complementary DNA was prepared from 100 ng total RNA by using one-cycle target labeling and a control reagent kit (Affymetrix). Hybridization and signal detection of HG-U133 Plus 2.0 arrays (Affymetrix) were performed. The microarray datasets of Gdeg^high and Gdeg^low cells were normalized and annotated with Expression Console™ Software (Affymetrix). Estimated gene expression levels were obtained as log2-transformed values. Genes were excluded from analysis if the detection call was “marginal” or “absent” in both Gdeg^high and Gdeg^low cells.

Genomic DNA was extracted from fresh cabbage leaves using cetyltrimethylammonium bromide according to a slightly modified published method (Murray and Thompson, 1980 (link)). The genomic DNA concentrations were determined using the NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and samples were then diluted to 40–50 ng/μL.
Polymerase chain reaction (PCR) experiments were conducted in 20-μL samples containing 4 μL DNA template (40–50 ng/μL), 2 μL 10 × PCR buffer (Mg²⁺ included), 1.6 μL dNTP (2.5 mM each), 0.8 μL forward and reverse primers (10 μM), 0.4 μL Taq DNA polymerase (2.5 U/μL), and 10.4 μL double-distilled H₂O. The PCR was completed in a GeneAmp PCR system 9700 thermal cycler (Life Technologies Co., Carlsbad, CA, USA) using the following program: 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s; 72°C for 10 min. The amplified products were separated by 8% (w/v) polyacrylamide gel electrophoresis (160 V for 1.5 h). The amplicons were visualized with silver nitrate staining (Brant et al., 1991 (link)).