RNA from MCs was extracted using Ribozol RNA Extraction Reagent (Amresco) as per the manufacturer’s recommendation, with 1 μg of RNA reverse transcribed into cDNA using qScript cDNA SuperMix Reagent (Quanta Biosciences). miRNA-enriched cDNA was generated using the qScript microRNA Quantification System (Quanta Biosciences). Quantitative real-time PCR was carried out using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) on the Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). mRNA and miRNA expression and fold changes were calculated using the ΔΔCT method, where 18S was used as a control for mRNA and U6 snRNA as a control for miRNA. Primers sequences used in the study are provided in Supplementary table 5 .
Ribozol rna extraction reagent
Manufactured by Avantor
Sourced in United States, United Kingdom
RiboZol RNA extraction reagent is a reagent used for the isolation and purification of total RNA from various biological samples, such as animal tissues, cell cultures, and body fluids. It is designed to provide efficient and reliable RNA extraction while maintaining the integrity of the extracted RNA.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Iq sybr green supermix, by Bio-Rad (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Onestep rt pcr kit, by Qiagen (3 mentions)
Abi 7900ht qpcr system, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Iscript reverse transcription supermix, by Bio-Rad (3 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Applied biosystems viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qscript cdna supermix reagent, by Quanta Biosciences (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (2 mentions)
Cfx manager 3, by Bio-Rad (2 mentions)
Cfx96 real time system, by Bio-Rad (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Iscript, by Bio-Rad (2 mentions)
Moloney murine leukaemia virus reverse transcriptase enzyme, by Promega (2 mentions)
69 protocols using ribozol rna extraction reagent
1
RNA Extraction and Quantification Protocol
2
Transcriptome Analysis of Rumen Ciliate Eudiplodinium caudatum
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Cells of E. caudatum MZG-1 were collected from a clonal monoculture of E. caudatum that was initially established from a single cell isolated from the rumen of gerenuk [36 (link)]. It was kindly given to us by Dr. Dehority (deceased). This monoculture does not have detectable fungus. Frozen stock cultures of E. caudatum MZG-1 were cryopreserved at − 80 °C and have been used in a number of studies [9 (link), 69 (link), 74 , 75 (link)]. The E. caudatum MZG-1 monoculture was fed a mixed feed containing ground wheat grain, ground alfalfa, and ground grass hays and maintained in SP medium [9 (link)]. The feeding and transfer procedures were conducted under a continuous stream of CO2 to protect the ciliate cells from exposure to oxygen. Total RNA was isolated from an actively growing E. caudatum MZG-1 monoculture after six hours of incubation at 39 °C after transferring to fresh SP medium containing the mixed feed. Total RNA was extracted using the Ribozol RNA extraction reagent (Amresco, Inc., Solon, OH) and then cleaned up using the RNeasy® mini kit according to the manufacturer’s instructions (Qiagen, Inc., Valencia, CA). mRNA was enriched using the Oligo Direct mRNA Mini Kit (Qiagen). One library was constructed for 2 × 100 paired-end sequencing from the mRNA and then sequenced following the manufacturer’s protocol on an Illumina HiSeq 2000 system.
3
Blood Sample Collection and Processing
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Blood samples (5 ml) were withdrawn 2 times during the study: 1- at the time of diagnosis (pre-therapy), 2- after receiving therapy (post-therapy such as surgery, chemotherapy or radiotherapy). Blood samples (2 ml) were allowed to clot at room temp for 45 min and centrifuged at 3,000 rpm for 5 min. Buffy coat was removed and serum was collected in microcentrifuge tubes (MCT) and kept at −80 °C until use.
Three milliliters of blood sample was taken in heparin coated vacutainers for real time quantitative polymerase chain reaction (RT-qPCR) analysis. After withdrawing blood, vials were kept on rocker for 30 min to maintain the room temperature, PBMC were isolated using Histopaque (Sigma-Aldrich, USA) density gradient centrifugation method following manufacturer’s instructions. Isolated PBMCs were used for total RNA isolation through Trizol method using RiboZol RNA extraction reagent (Amresco, USA). The purity, integrity, and quantification of the RNA samples were analyzed using the Bioanalyser (Agilent Technologies, USA).
Three milliliters of blood sample was taken in heparin coated vacutainers for real time quantitative polymerase chain reaction (RT-qPCR) analysis. After withdrawing blood, vials were kept on rocker for 30 min to maintain the room temperature, PBMC were isolated using Histopaque (Sigma-Aldrich, USA) density gradient centrifugation method following manufacturer’s instructions. Isolated PBMCs were used for total RNA isolation through Trizol method using RiboZol RNA extraction reagent (Amresco, USA). The purity, integrity, and quantification of the RNA samples were analyzed using the Bioanalyser (Agilent Technologies, USA).
4
Quantitative Analysis of Kidney Injury Markers
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The whole ischemia-reperfusion treated left kidneys were used to obtain mRNA with a RiboZol RNA extraction reagent (AMRESCO, VWR life science, Radnor, PA, USA). Reverse transcription was done with a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Quantitative real-time PCR was performed using the ready-to-use mix of enzyme and SYBR Green qEvaGreen (qARTA Bio, Inc., Carson, CA, USA) in a Lightcycler 96 analyzer (Roche Molecular Systems, Inc., Pleasanton, CA, USA). The expression of kidney injury molecule 1 (KIM-1), IL-6, CD31, HIF-1α, VEGF, VEGF-R2 and CTD genes in the ischemia-reperfusion treated left kidney was determined using specific oligonucleotides (Table 1 ). The amplification conditions were as follows: 10 s at 95 °C, 30 s at primer-specific annealing temperature and 30 s at 72 °C for 40 cycles. Relative expression for each gene was quantified using the 2-ΔΔCt threshold method. Hypoxanthine phosphoribosyl transferase (HPRT) mRNA was used as a housekeeping gene.
5
Analyzing Transgenic Cucumber Plant RNA
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RNA from the leaves of wild (control) and transgenic cucumber plants was isolated using RiboZol™ RNA Extraction Reagent (Amresco) and resolved on 1% denaturing agarose gel. One microgram of isolated RNA was used for the cDNA synthesis via reverse transcriptase cDNA synthesis kit (Thermo Fischer Scientific). One microliter of cDNA was used as template for the polymerase chain reaction using primers specific to E-cassette as per the conditions described in Table 1 . The resultant amplified products were resolved on 1% agarose gel using electrophoresis.
6
Molecular Mechanism of Ellagic Acid Inhibition
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The small molecule inhibitor, ellagic acid dihydrate (TBBD), CARM1 polyclonal antibody (09-818), anti-actin polyclonal antibody (A2066), and salmon calcitonin (05232401) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sepharose Protein A was obtained from Rockland Immunochemicals, Inc. (Gilbertsville, PA, USA). VDR (C-20) and OPN (P-18) antibodies and anti-rabbit (sc-2004) and anti-goat (sc-2020) horseradish peroxidase-(HRP) conjugated) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Asymmetric dimethyl H3R17 antibody (ab8284) was obtained from Abcam (Cambridge, MA, USA). Donkey anti-rabbit IgG HRP (711-035-152) was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). 1,25(OH)2D3 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Pre-stained protein markers and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad. The luciferase assay system (E1501) was purchased from Promega (Madison, WI, USA). RiboZol RNA Extraction Reagent was purchased from Amresco (Solon, OH, USA).
7
Quantification of PPARβ/δ Target Genes
8
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with Ribozol RNA extraction reagent (Amresco). One μg of RNA was reverse-transcribed using the high capacity RNA-to-cDNA kit (Life Technologies). qPCR was performed with SYBR Green JumpStart Tag ReadyMix (Sigma) according to the manufacturer's protocol using ABI StepOne Plus Real-Time PCR System (Applied Biosystems, 4376600). Relative mRNA levels were determined using 2−ΔCt method. The GAPDH gene was used as an internal control. The primer sequences were taken from Kondaveeti et al. [20 (link)] and their specificity confirmed by melting curve analysis (data not shown). All primer pairs were synthesised by Sigma.
9
Quantitative RT-PCR Analysis of Neurological Markers
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Total RNA was purified from homogenized brain specimen using RiboZol RNA extraction reagent (Amresco, Solon, USA) following the manufacturer's instructions. Isolated total RNA was used as template for reverse transcriptase-polymerase chain reaction (RT-PCR) using OneStep RT-PCR Kit (Qiagen, UK). Sequences of the primers used for PCR amplification were as follows: Ngb forward; 5-CTCTGGAAC ATGGCACTGTC-3; reverse, 5-GCACTGGCTCGTCTCTTACT-3; product size 425 bp, for S100B forward; 5-TTGCCCTCATTGATGTCTTCCA-3 and 5-TCTGCCACGGGGAAACGGCTCA-3; product size 296 bp, and for 18s; forward, 5-TTGACGGAAGGGCACCACCAG-3, reverse, 5-GCACCACCACCCACGGAATCG-3; product size 131 bp. RT-PCR products were separated on 2% agarose gel, visualized by ethidium bromide staining. The intensity of the PCR product bands was quantified using gel documentation system software (Biometra GmbH, Germany).
10
Transcriptomic Analysis of Cellular RNA via RNA-Seq
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