Nanodrop

Total RNA (tRNA) was extracted from the control and CRTAC1 challenged cells using a Nucleospin RNA Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. Cells were collected directly into the kit lysis buffer supplemented with β-mercaptoethanol and homogenized manually by pipetting up and down. A DNase I digestion protocol was performed directly on the Nucleospin columns during RNA extraction to eliminate contamination with genomic DNA. Total RNA was eluted in 40 µL of MilliQ water and quantified using a Nanodrop (Nanoquant, TECAN, Männedorf, Switzerland). The integrity of the total RNA was checked by running samples on a 1% TAE agarose gel. All samples were stored at −80 °C until use.
For cDNA synthesis, 500 ng of DNAse treated tRNA was used and cDNA synthesis was performed using a PrimeScript-RT reagent kit (Takara Bio Inc, Kusatsu, Shiga, Japan), following the manufacturer’s instructions. The quality and quantity of the cDNA synthesis was tested by PCR amplification of the human ribosomal subunit 18S rRNA (Table 1) using the following cycles: 95 °C for 3 min, followed by 25 cycles of 95 °C for 20 s, 58 °C for 20 s, 72 °C for 20 s and a final extension step of 72 °C for 5 min. The amplified PCR products were visualized on a 2% TAE agarose gel.

Quality control (QC) for prepared genomic DNA was performed; A_260/280 > 1.6 and A_260/230 > 1.6 for UV absorbance using Nanodrop (Tecan) and ≥5 ug fluorescence concentration using the Quant-iT BR assay kit (Invitrogen). DNA was run on 0.7 % agarose gels to assess degradation.
Genomic DNA was fragmented into approximately 100–300-bp insert sizes by sonication. Each DNA fragment was end-repaired using the Paired-End DNA Sample Prep Kit (Illumina; San Diego, CA, USA), followed by the addition of a 3′-A overhang and ligation of the adapters. Size-selection for DNA fragments of 200–300 bp was performed using a gel extraction kit (Qiagen). The constructed DNA samples were quantified using Quant-iT™ dsDNA High Sensitivity Assay Kit (Invitrogen; Carlsbad, CA, USA) on an Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA). After qPCR validation, the resulting libraries were subjected to paired-end sequencing with a 100-bp read length using the Illumina HiSeq 2500 platform (Illumina). Raw image files were processed by Illumina Real-Time Analysis (RTA) for image analysis and base calling.

Survey slides of the biopsies were examined and the carcinoma cells identified. Since all material was reevaluated by an experienced uro-genito-pathologist to ensure that only tumor tissue was included. Subsequently, slides were stained with cresyl-violet 1% and the carcinoma cells were laser micro dissected using the PALM laser microbeam system. RNA extraction was performed using RNeasy® Micro kit from Qiagen (Germany). Flow through from the RNA extraction contains RNA fragments that are shorter than 120 nucleotides in length, thus the total microRNA fraction was contained within the flow through. A second microRNA extraction was performed on the flow through with RNeasy micro kit optimized for extracting micro RNAs.
RNA extraction from FFPE tissue was performed using RNeasy® FFPE Kit (Qiagen Germany) in which the total RNA fraction contains RNA fragments down to 18 nucleotides in length. RNA260/280 ratio and the RNA concentration of each sample was measured using NanoDrop (Tecan).

IPF lung-derived or normal lung fibroblasts as well as normal primary lung epithelial and endothelial cells (Lonza) were cultured and expanded using standard cell culture techniques. In this study, the cells were treated with esomeprazole or vehicle for 24 h in the presence or absence of bleomycin (Sigma; at 25 µg/mL final concentration). Subsequently, the cells were harvested and total RNA was extracted using the PerfectPure RNA Cell and Tissue kit (5 PRIME). Next, the concentration and quality of the RNA were validated using a Nanodrop (Tecan) and 2 µg of RNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). The resulting cDNA was used for gene expression study by quantitative RT-PCR. Quantitative RT-PCR (qRT-PCR) was performed using standard TaqMan gene expression assay using proprietary “best coverage” primer/probe sets (Life Technologies) as described below.

Total RNA (approximately 1 µg of RNA per sample) was isolated from brain tissues 3 days after hypoxia/ischemia using the RNeasy Mini Kit (Qiagen, Valencia, CA). The quantity of RNA was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Tecan NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA-Reverse Transcription Kit (Thermo Fisher Scientific, USA). The reverse transcription reaction and quantitative polymerase chain reaction (qPCR) were run on the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) as previously described [21 (link)]. Amplification was performed in a total volume of 20 µl containing 10 µl of TaqMan Gene Expression Master Mix and 1.0 µl of reverse transcription product as the PCR template. A standard qPCR procedure was utilized: 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was set during the exponential phase, and the delta Ct method was used for data analysis. To evaluate reference gene expression, the RefFinder web-based comprehensive tool was used [22 (link)]. For our study, NormFinder, BestKeeper and delta Ct recommended Hprt as the most stable reference gene.

miRNA (approximately 300 ng of RNA per sample) was isolated from the brains of 10-day-old rat pups subjected to HI and/or treated with 3,3’-diindolylmethane (10 mg/kg) using the miRNeasy Mini Kit (Qiagen, Valencia, CA). The quantity of RNA was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Tecan NanoDrop, USA). First-strand cDNA synthesis (optimized for 20 ng of RNA) was performed using the miRCURY LNA RT Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Quantitative polymerase chain reaction (qPCR) was conducted with the miRCURY SYBR Green PCR Kit and miRCURY LNA miRNA PCR custom-made assays (Qiagen, Valencia, CA). The reverse transcription reaction and qPCR were run on the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) as previously described [23 (link)]. The threshold value (Ct) for each sample was set during the exponential phase, and the delta-delta Ct method was used for data analysis. The U6 coding gene was employed as a reference gene based on the recommendations of the statistical tools of BestKeeper, delta-Cq, and NormFinder.

At 10 h after MSC transplantation, the livers, lungs, and hearts were isolated to investigate the presence of the transplanted cells in these organs. Depending on the weight of the organ, lysis buffer was added and homogenized. Afterwards, DNA was extracted using a genomic DNA extraction kit (Bioneer Corp, Daejeon, South Korea) and quantified using a spectrophotometer (Nanodrop; TECAN). Real-time PCR (RT-PCR) was performed using 25 ng DNA, 5 μL SYBER green (Thermo Scientific, Waltham, MA, USA), 37.5 nM ALU primer, and 450 nM GAPDH primer. The primer sequences used were as follows: ALU, 5′-GTCAGGAGATCGAGACCATCCC-3′ (F) and 5′-TCCTGCCTCAGCCTCCCAAG-3′ (R), and GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (F) and 5′-TCCACCACCCTGTTGCTGTA-3′ (R). Calibration curve was prepared using crossing point (Cp) values obtained with various amounts of human DNA (16%, 8%, 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.0156%, 0.0078%, and 0.0039%).