Gene elute mammalian total rna miniprep kit

RNA extraction was performed from cell cultures at day 15 and 24 after start of differentiation to CMs in accordance with the manufacturer’s protocols using GeneElute Mammalian Total RNA Miniprep kit (Sigma-Aldrich, Cat No. RTN70) including on-column DNA digestion. RNA was converted to cDNA using the SuperScript III First-Strand Synthesis SuperMix-kit (Invitrogen, Cat No. 18080400). qPCR was performed using the Platinum SYBR Green qPCR SuperMix-UDG-kit (Cat No. 11733038) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the following PCR protocol: 2 min 50 °C, 2 min 95 °C followed by 40 cycles of 20 s 95 °C and 45 s 60 °C on 10 ng cDNA per 20 µl reaction. Primers were used at a final concentration of 200 nM (Supplementary Table 2). Analysis of qPCR results was performed using CFX Manager-software (Bio-Rad).

Retinal tissue was homogenized by trituration using a pipette (n = 5/group). The RNA isolation of the retina was carried out according to the manufacturer's introduction using the Gene Elute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). For total RNA isolation of optic nerve tissue, the ReliaPrepTM RNA Tissue Miniprep System (Promega, Madison, WI, USA) was taken. Prior to isolation, optic nerve tissue was incubated in liquid nitrogen and then homogenized with a pestle (n = 5/group). An additional DNase digestion at RT for 15 min ensured that no genomic DNA contaminated RNA was obtained. The concentration and purity of the isolated RNA was determined photometrically using the BioSpectrometer® (Eppendorf, Hamburg, Germany). One microgram RNA and random hexamer primers were applied for reverse transcription using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). RT-qPCR experiments were done with SYBR Green I in a Light Cycler 96® (Roche Applied Science, Mannheim, Germany). For each primer pair (Table 4), efficiencies were determined by a dilution series of 5, 25, and 125 ng cDNA. Expression in retina and optic nerve tissue was normalized against the housekeeping genes β-actin (Actb) and 18S ribosomal RNA (Rn18S), respectively.

Total RNA from cultured cells was isolated using Gene Elute Mammalian total RNA Miniprep Kit (Sigma) according to the provided protocol including DNase digestion with the RNase-free DNase Set (Qiagen). Isolation of genomic DNA from cultured cells was performed using Quick-gDNA MiniPrep Kit (Zymo Research) according to the provided protocol. Concentration of RNA and DNA was determined by using a ND-1000 spectrophotometer (NanoDrop Technologies) using ND-1000 V3-8.4 software.

RNA was harvested from transfected myofibroblasts at several passages using the GeneElute MammalianTotal RNA Mini-prep Kit (Sigma-Aldrich), per manufacturer’s instructions.
cDNA was synthesized and mRNA expression of the following genes was evaluated: α-SMA (forward: CTG TTC CAG CCA TCC TTCAT; reverse: CCG TGA TCT CCT TCT GCA TT), vimentin (forward: TGT CCA AAT CGA TGT GGA TGT TTC; reverse: TTG TAC CAT TCT TCT GCC TCC TG), hTERT (forward: TGGTTTCTGTGTGGTGTCA; reverse: TTGTCGCCTGAGGAGTAGA), TRPV1 (forward: GAC TTC AAG GCT GTC TTC ATC ATC C; reverse: GAC TTC AAG GCT GTC TTC ATC ATC C), TLR4 (forward:CAG GGC TTT TCT GAG TCG TC; reverse TGA GCA GTC GTG CTG GTA TC). Human 18s (forward CCA TGA AGA GGT GAG CGG GGA TTG; reverse ATT AAG TCC CTG CCC TTT GTA CAC) was used as the endogenous control to normalize samples using the ΔΔCT method of relative quantitation, where CT is the threshold cycle. Expression data is presented in log form. Primary esophageal myofibroblasts were used as controls.

Quantitative PCR (qPCR) was performed as previously described [19 (link), 31 (link)]. Total RNA was isolated from mammary and uterus tissues using the GeneElute Mammalian Total RNA Miniprep Kit (Sigma, Rehovot, Israel) combined with on-Column DNase I Digestion Set (Sigma, Rehovot, Israel).
Reverse transcription was performed using qScript cDNA Synthesis Kit (Quanta BioSciences, Gaithersburg, MD, USA) and cDNA was used for subsequent qPCR reactions. Quantitative RT–PCR was conducted on a StepOne Plus PCR instrument (Applied Biosystems) using the FAST qPCR Universal Master Mix (Kappa Biosystems, Boston, MA, USA). PCR primers used in this study are listed in Supplementary Table 2. All reactions were performed in triplicates and the gene expression levels for each amplicon were calculated using the ΔΔCT method and normalized against HSP90 mRNA.

The GeneElute Mammalian Total RNA Miniprep Kit (Sigma) was used to extract RNAs, following the manufacturer’s protocol. Briefly, 1 μg of RNAs was retrotranscribed using the SuperScriptTM III First-Strand Synthesis SuperMix Kit (Invitrogen) and diluted in DEPC water. Primers (Supplementary Table S1) for RT-qPCR were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/) and tested for their efficiency. RT-qPCR was performed, according to the SYBR Green Mix (Invitrogen) protocol, on real-time system Realplex2 Master Cycler (Eppendorf) or on ViiA7 Real-Time PCR system (Invitrogen).