Nanodrop nd 2000

Genomic DNA was extracted from fresh frozen samples by using Qiagen Blood and Cell culture DNA kit. The extracted DNA yield and quality were assessed using Nanodrop ND2000 (Thermo scientific). The extracted DNA (about 1 μg) from the fresh‐frozen tissue specimens were sent to Genotypic Technology Pvt Ltd, Bangalore for exome sequencing. Genomic DNA from FFPE blocks was extracted using Qiagen QiAmp DNA FFPE Tissue kit as per manufacturer instructions. The extracted DNA yield and quality were assessed using Nanodrop ND2000 (Thermo scientific). These samples were further checked for integrity by PCR amplification of GAPDH (96 bp). These samples were used for extended Sanger validation of identified variants in exome sequencing.

For gene expression analysis, samples were homogenized in RNEasy Plus Universal (Quiagen, Hilden, Germany) reagent with a tissue grinder, RNA was extracted according to the manufacturer’s instructions, and the RNA concentration was determined using a spectrophotometer (Nanodrop ND-2000, Fisher Scientific, Schwerte, Germany). One microgram of RNA from each sample was reverse transcribed into cDNA using Transcriptor First Strand Synthesis Kit (Roche, Mannheim, Germany) and cDNA amplification was performed using a Biorad CFX 96 real-time polymerase chain reaction system with intron-spanning primers and SYBR Green Reaction Mix (Agilent, Santa Clara, CA, USA). The relative gene expression was calculated using the delta-delta-Ct algorithm (DDCT method). Each sample was normalized to the average expression of the three housekeeping genes: Receptor expression-enhancing protein 5 (REEP-5), vacuolar protein sorting protein (VPS-29), Proteasome subunit beta type-4 (PSMMB-4). Expression levels of the cartilage-specific markers Sox 9, Collagen II, and MIA, and the hypertrophy-related marker, Collagen-type X and the fibrous-tissue related marker Collagen I, were determined and relative gene expression compared to mean gene expression of housekeeping genes was reported. For complete sequences of all primers see Table A1.

The choroid plexus was rapidly dissected and placed into RNAlater Stabilization Solution (Ambion). Total RNA was purified using GeneJet RNA purification kit (Thermo Fisher Scientific). The concentration of purified RNA was determined by absorbance at 260 nm using a NanoDrop ND‐2000 (Fisher Scientific). Then 80–120 ng of RNA was reverse transcribed using iScript Reverse Transcription Supermix (BioRad). qPCR amplification was performed with Step One Plus real time PCR system (Applied Biosystems, Foster City, CA, USA) using a commercially available TaqMan Gene Expression Assay (Slc4a5: Mm01190997_m1, control – Actb: Mm00607939_s1; Applied Biosystems) and the universal TaqMan Gene Expression Master Mix. PCR cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The results are presented as the relative NBCe2 expression fold‐change (2^−ΔΔCT method) compared to the calibrator: the RLuc siRNA‐injected mice.

The purity of the collected renal tubules and the expression of NBCe2 was determined by RT-PCR (see Supplementary Table S1 for primer sequences and product sizes). RNA from the tubules was purified using an RNeasy micro kit (Qiagen, Copenhagen, Denmark) according to the manufacturer’s instructions. The concentration of purified RNA was determined by absorbance at 260 nm using a NanoDrop ND-2000 (Fisher Scientific) and 20–40 ng of RNA was reverse transcribed by iScript Reverse Transcription Supermix (Bio Rad, Copenhagens, Denmark). PCR amplification was performed for each transcript by mixing cDNA with 1 pmol of primers and 5× HOT FIREPol Blend Master Mix (Solis BioDyne). The PCR reaction was performed for 35 cycles after 15 min at 95°C: denaturation was performed for 30 s at 95°C, annealing at 60°C for 30 s, and elongation at 72°C for 1 min. PCR products were mixed with 2 μl of DNA Gel Loading Dye (6×; Thermo Scientific) containing 0.05% 10000 × GelRed (Biotium, BioNordika Denmark, Herlev, Denmark) nucleic acid gel stain, separated by 1% agarose gel electrophoresis, and photographed under ultraviolet illumination.

The total RNA was prepared from the brain using QIAzol Reagent, cleaned with RNeasy Mini kit (Qiagen; Milano- Italy), and eluted in RNase free water. The purity and concentration of RNA were evaluated by spectrophotometry using NanoDrop ND-2000 (ThermoScientific, Milano, Italy). Complementary DNA (cDNA) synthesis was performed using RT² First Strand kit (Qiagen) following the manufacturer’s instructions. The expression analysis of 84 genes associated with hypoxia was carried out with the RT² Profiler™ PCR Array Rat Hypoxia Signaling Pathway (PARN-032Z, Qiagen) according to the manufacturer’s guidelines in 96-well plates with a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). The raw data obtained was uploaded into GeneGlobe for analysis. Relative quantification of messenger RNA (mRNA) expression was calculated using the comparative cycle threshold (CT) method and expressed as log2-fold change of expression. The fold change (2^−∆∆Ct) is the normalized gene expression (2^−∆Ct) in the test sample which is the ipsilateral hemisphere divided by the normalized gene expression (2^−∆Ct) in the control sample (the contralateral hemisphere).

We chose E40, E50, and E60 stage corresponding to the cap stage, early bell stage, and late bell stage mandibular third deciduous molar (Dm3), respectively. The Dm3s were harvested from the staged embryo mandible under a stereomicroscope (LEICA), rinsed in sterile phosphate buffered saline (PBS), placed into 150 μL RNAlater stabilization solution (ThermoFisher Scientific) for 16 h at 4°C, and then stored at −20°C. Total RNA from Dm3 at each stage was extracted separately using a miniKit (QIAGEN, Germany) according to the manufacturer's protocol. RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, USA). Total RNA was then quantified on a NanoDrop ND-2000 (Thermo Scientific, USA). The RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA), and the RNA with RIN (RNA integrity number) > 8.0 is acceptable for transcriptome library construction. [17 , 18 (link)]