Aurum total rna fatty and fibrous tissue kit

Ileum samples from the WT mice were removed to determine the expression of MyD88, Tlr2, Tlr9, Myd88 and Traf6. In addition, intestinal samples of MyD88^-/-, TLR2^-/- and TLR9^-/- mice were removed to quantify the expression of cyclooxygenase–2 (Cox–2) and IL–18. Total RNA isolation was performed using the Aurum^TMTotal RNA Fatty and Fibrous Tissue Kit (Bio-Rad, CA, USA). The yield and quality of total RNA were determined spectrophotometrically using 260 nm and a 260/280-nm ratio, respectively. One microgram of total RNA from the intestinal samples in a final volume of 20 μl were reverse-transcribed into cDNA in the C1000 Touch^TM Termal Cycler system with the iScript^TM cDNA synthesis kit from Bio-Rad. Real-time quantitative PCR analysis of the mRNA was performed in an CFX96 Touch^TM real-time PCR detection system instrument from Bio-Rad using the iQTM SYBR® Green Supermix (Bio-Rad, CA, USA) as indicated by the manufacturer. All samples were run in duplicate, and the relative mRNA expression level was determined after normalizing all values to those of β-actin. All samples were evaluated for the dissociation characteristics of the double-stranded DNA during heating (melting curve analysis). The relative gene expression was determined using the 2^-ΔΔCt method [34 (link)] with β-actin as the housekeeping gene. The primer pairs used in this study are shown in Table 1.

Total maize RNA was extracted from the ground kernel samples with the Aurum^TM Total RNA Fatty and Fibrous Tissue Kit from Bio-Rad (Bio-Rad Laboratories, Hercules, CA, United States). The extraction followed the manufacturer’s protocol with the following minor modifications. The ground kernel samples were placed into sterile 2 mL tubes with 100 mg per sample. 1 mL of cold Trizol was added to each sample and vigorously vortexed to ensure a complete suspension of kernel powder. The resulting lysate was incubated at room temperature for 5 min. The tubes containing the lysate were centrifuged at 12,000 rpm for 5 min at 4°C. The Aurum^TM Total RNA Fatty and Fibrous Tissue Pack instructions were followed from this point forward.

Samples were homogenized using an ultrasound homogenizer (Sonoplus HD3100, Brandelin) in 1 ml PureZOL and total RNA was isolated via column fractionalization using the Aurum^TM Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad Laboratories Inc.; Hercules, CA, USA) following the manufacturer’s instructions. All the samples were DNAse treated using an on-column DNAse I contained in the kit to remove genomic DNA. The RNA quantity for each sample was analyzed in the NanoDrop 2000 Spectrophotometer (Thermo Scientific; Wilmington, DE, USA). BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories Inc.; Hercules, CA, USA) was then used to convert mRNA into cDNA, following the manufacturer’s instructions.
qPCR primers (Tataa Biocenter; Gothenburg, Sweden) were designed following the NCBI Sequence database, including the local factors chosen in order to characterize the immune, inflammatory, and bone metabolic pathways (Table 1 and Table 2). All primers had efficiency between 90% and 110%.

Total RNA was isolated from the samples using the Aurum^TM Total RNA Fatty and Fibrous Tissue kit, according to the manufacturer’s protocol (Bio-Rad Laboratories, Inc., Hercules, CA, United States). The total RNA quality was assessed by visualizing the integrity of the 18S and 28S rRNA after electrophoresis in 1% agarose gels. Total RNA concentration was determined via spectrophotometry (NanoDrop ND-1000 spectrophotometer; Nano-Drop Technologies, Waltham, MA, United States). RNA solutions were stored at −75°C.

First, total RNA of each sample was extracted using AurumTM Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, USA) and was quantified by NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). Reverse transcription was then demonstrated via iSciptTM cDNA Synthesis Kit (Bio-Rad, USA) by adding 1 μg of total RNA on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). Real-time PCR was performed by means of SsoFast™ EvaGreen® Supermixes (Bio-Rad, USA) on CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA) through adding an equal volume (2 μl) of cDNA products of each sample. The target genes were Slc2a4 (for Glut4; Bio-Rad, qRnoCID0001996, USA) and Acss2 (for AceCS1; Bio-Rad, qRnoCID0005949, USA), β-actin was selected as an internal control (forward primer sequence: 5′-CGGTCAGGTCATCACTATC-3′; reverse primer sequence: 5′-TGCCACAGGATTCCATAC-3′). Further ΔCq of the target gene expression was calculated and normalized with β-actin by the equation ΔCq= Cq(target) – Cq(β-actin).

RT-qPCR was used to assess gene expression of inflammatory markers in brain and muscle tissue. Mice were anesthetized with isoflurane and decapitated at either 24 h or 35 days post-injury. For the examination of brain tissue, the ipsilateral cortex directly under the impact site was rapidly dissected, frozen in liquid nitrogen, and stored at −80°C. For the examination of muscle tissue, 100 mg of tissue was rapidly dissected from the injection site in the right hamstring, frozen in liquid nitrogen, and stored at −80°C. Frozen brain and muscle tissue was homogenized with PureZOL^TM RNA isolation reagent, and total RNA were extracted using the Aurum^TM Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, USA) (36 (link)). RNA for each sample (brain = 600 ng, muscle = 1,000 ng) was then reverse transcribed to cDNA.
The primer sequences for the inflammatory genes of interest are summarized in Table 2 (30 (link), 37 (link)). qPCR was run using the iCycler iQ Multi-Color RT-PCR detection system (using SsoFast^TM EvaGreen, Bio-Rad, Hercules, USA). To establish specificity of DNA products, melt-curve analysis was performed. Cycle threshold (Ct) values were collected for analysis, and data was normalized to internal control (brain = Gapdh; muscle = β2-microglobulin). Relative quantification of genes of interest mRNA expression was determined using the 2^−ΔΔCt method (5 (link)).

Total RNA was isolated from 50 mg muscle tissue (triceps brachii) of case 2 and an unrelated Maine coon cat without signs of muscular dystrophy using the AurumTM Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s spin protocol instructions. Subsequent cDNA synthesis was performed as described by Van Poucke et al. [22 (link)] and 2 µL was used as a PCR template.