Frozen ASC-seeded tendon scaffolds were homogenized using a Tissue Lyser II (Qiagen, Hilden, Germany) and samples were treated with proteinase k (Qiagen) at 55 °C. Total RNA of scaffold-seeded and monolayer ASC was then isolated using the RNeasy® Mini Kit with On Column DNase digestion (Qiagen). RNA was converted to first strand complementary DNA (cDNA) using Reverse Transcriptase RevertAid H Minus (ThermoFisher Scientific). Next, cDNA was mixed with the respective primers and iQ™ SYBR Green Supermix (Bio-Rad Laboratories, Munich, Germany). Primer sequences for target and housekeeping genes are given in Table 3 . Relative quantification of cDNA was performed using an Applied Biosystems™ 7500 Real Time PCR System, and gene expression ratios were calculated according to [56 (link)]. Additionally, for further comparisons between groups as well as for graphical presentation of data, and gene expression was normalized to the corresponding controls within each group (monolayer, static, and dynamic scaffold cultures without inflammatory stimulation).
Rneasy mini kit with on column dnase digestion
Manufactured by Qiagen
Sourced in Germany
The RNeasy Mini Kit with on-column DNase digestion is a laboratory product for the purification of total RNA from various sample types. It provides an efficient method for the isolation of high-quality RNA, including the removal of genomic DNA during the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Tissuelyser 2, by Qiagen (1 mentions)
Revertaid h minus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Proteinase k, by Qiagen (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Primepcr plate, by Bio-Rad (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Cyp8b1, by Bio-Rad (1 mentions)
Rnaprotect bacteria reagent, by Qiagen (1 mentions)
Agilent 8 60k array, by Agilent Technologies (1 mentions)
Feature extraction version 10, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Low input quick amp wt labeling kit, by Agilent Technologies (1 mentions)
Hybridization oven, by Agilent Technologies (1 mentions)
4 protocols using rneasy mini kit with on column dnase digestion
1
Quantitative Analysis of ASC Gene Expression
2
Quantitative Real-Time PCR Analysis
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PrimePCR for Nr0b1, Cyp7a1 and Cyp8b1 was purchased from Bio-Rad (California, USA). Gene-specific primers were added in a pre-made PrimePCR plate from Bio-Rad (Solna, Sweden). Total RNA in freeze-dried liver tissues (approximately 30 mg) was purified using the RNeasy Mini Kit with on-column DNase digestion (Qiagen GmbH, Hilden, Germany), and converted to cDNA using the Thermo Scientific™ RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The quantitative real-time PCR was performed using a Bio-Rad T100 Thermal Cycler with SYBR Green SsoAdvanced Universal Supermix (Bio-Rad, California, USA). The amplification protocol was as followed: activation at 95 °C for 2 min, followed by 40 cycles of 5 s denaturation at 95 °C, and annealing at 60 °C for 30 s. Specificity of the PCR products was checked by performing a melt curve analysis. GAPDH was used as a reference gene. The ∆∆Ct method was used to calculate relative mRNA expression.
3
Transcriptome analysis of EPEC and EHEC
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Quadruplicate overnight cultures of WT and Δler strains, grown in LB broth (Miller formulation) were diluted 1/100 into DMEM buffered with 25 mM HEPES and incubated at 37°C, with aeration by shaking at 200 rpm (i.e. inducing conditions for expression of the LEE). Samples were harvested at mid and late log phases of growth (OD600 of 0.4 and 0.9 for EPEC; 0.5 and 1.1 for EHEC). Messenger RNA was stabilized immediately by pipetting the samples directly into RNAprotect Bacteria reagent (Qiagen) before purification of total RNA using the RNeasy Mini Kit with on-column DNase digestion (Qiagen).
4
Transcriptome Analysis of E. coli Using Microarray
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The cells were cultured for 16–19 h and then sampled at the time of the logarithmic growth phase (OD600 values were 0.072–0.135). Aliquots of the cells were immediately added to the same volume of ice-cold ethanol containing 10% (w/v) phenol. RNA extraction was performed using a RNeasy mini kit with on-column DNase digestion (Qiagen), following the manufacturer’s protocol. The purified RNA was quality-controlled using an Agilent 2100 Bioanalyzer and an RNA 6000 Nano kit (Agilent Technologies). A microarray experiment was performed using an Agilent 8 × 60 K array, which was designed for the E. coli W3110 strain so that 12 probes were contained for each gene. Purified total RNA (100 ng) was labeled with Cyanine3 (Cy3) using a Low Input Quick Amp WT labeling kit (One-color; Agilent Technologies). The Cy3-labeled cRNA was checked for its amount (>5 µg) and specific activity (>25 pmol/µg) using NanoDrop ND-2000. Then, the cRNA of 600 ng was fragmented and hybridized to a microarray for 17 h at 65°C, rotating at 10 rpm in a hybridization oven (Agilent Technologies). The microarray was then washed and scanned according to the manufacturer’s instructions. Microarray image analysis was performed using Feature Extraction version 10.7.3.1 (Agilent Technologies). The resulting gene expression levels were normalized using quantile normalization.
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