Snap-frozen mouse organs (kidney, liver, brain, lung, spleen, heart, prostate, and testis) were powderized and RNA was isolated using TRI Reagent (RNA/DNA/Protein isolation reagent, Molecular Research Center Inc., Ohio, U.S.A.) according to the manufacturer’s instructions. RNA (1 µg) was used to produce cDNA (High Capacity RNA-to-cDNA Kit, Applied Biosystems, Life Technologies, Thermo Fisher Scientific Inc.) according to manufacturer’s instructions. The cDNA was then used as template in qPCR (LightCycler, Roche) with enzyme mix (SYBR Green/ROX qPCR Master Mix (2×), Thermo Scientific, Thermo Fisher Scientific Inc.) and specific primers (ODCrp: forward primer 5′-ACACACCTGAGAGCTACAGA and reverse primer 5′-TCCTGGATCTAGGGAAGACT, β2M: forward primer 5′-ATGTCTCGATCCCAGTAGAC and reverse primer 5′-GCTATCCAGAAAACCCCTCA). Sample quantitations were normalized using the invariant endogenous control β2M. Finally, the results (mean + S.D.) of three biological replicates were scaled to the result of untreated male control.
Tri reagent rna dna protein isolation reagent
Manufactured by Molecular Research Center
Sourced in United States
TRI Reagent is a versatile reagent for the isolation of RNA, DNA, and protein from a variety of biological samples. It is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components that facilitate the separation of the cellular components during the isolation process.
Automatically generated - may contain errors
Lab products found in correlation
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler, by Roche (1 mentions)
Light cycler pcr system, by Roche (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Mircute plus mir first strand cdna kit, by Tiangen Biotech (1 mentions)
Mircute plus mirna qpcr kit sybr green, by Tiangen Biotech (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hiscript 3 1st strand cdna synthesis kit gdna wiper, by Vazyme (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Sensimix sybr fluorescein kit, by Meridian Bioscience (1 mentions)
Myiq real time pcr detection system, by Bio-Rad (1 mentions)
Oligo dt 18 primer, by Meridian Bioscience (1 mentions)
M mlv reverse transcriptase, by New England Biolabs (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
7 protocols using tri reagent rna dna protein isolation reagent
1
Quantification of Gene Expression in Mouse Organs
2
Quantifying MCP-1 and CX3CL1 Gene Expression
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The gene expression levels of MCP-1 and CX3CL1 were assessed by real-time PCR. Total RNA was isolated using the TRI reagent (RNA/DNA/protein isolation reagent, Molecular Research Center, Cincinnati, OH, USA) and reverse-transcribed into cDNA using a random hexamer and M-MLV reverse transcriptase (Promega, Madison, WI, USA) at 23°C for 10 minutes, 42°C for 60 minutes, and 95°C for 5 minutes. The cDNA was incubated with SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) containing 10 pg/mL of forward and reverse primers, and amplified using the Light Cycler PCR system (Roche Applied Sciences, Indianapolis, IN, USA). RT-PCR was performed using the following rat-specific primers for MCP-1: 5’-ACGTGCTGTCTCAGCCAGAT-3’ (forward) and 5’-GTTCTCCAGCCGACTCAT TG-3’ (reverse), CX3CL1: 5’-CACAAGATGACCTCGCCAAT-3’ (forward) and 5’-GCTGTCTCGTCTCCAGGATG-3’ (reverse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5’-GCTCTCTGCTCCTCCCTGTT-3’ (forward) and 5’-CACACCGACCTTCACCATCT-3’ (reverse). For PCR amplification, the cDNA was amplified by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds. During the first cycle, the 95°C step was extended to 3 minutes. GAPDH was amplified in the same reaction as the reference gene.
3
Quantitative Gene Expression Analysis
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Approximately 100 mg of leaf tissue was homogenized in liquid nitrogen, and total RNA was isolated and purified using TRI-REAGENT® RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) and 0.5 µg of total RNA incubated first at 37 °C for 5 min for the DNase reaction and second at 37 °C for 15 min for the reverse transcriptase reaction. Real-time PCR was performed using a CFX Connect Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primers (Table S2 ). The following protocol was used: initial polymerase activation: 60 s at 95 °C; 40 cycles of 15 s at 95 °C and 30 s at 60 °C; and then melting curve analysis preset by the instrument was performed. Relative transcript abundances were determined after normalizing raw signals to the transcript abundance of a housekeeping gene (actin). Samples and data were excluded if abnormal quantification cycle (Cq) values for the actin gene were obtained.
4
Quantitative Analysis of miR-146a-5p in Caco-2 Cells
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A total of 5x105 cells/well was planted in 48 well cell culture plate, cultured at 37˚C for 24 h, total RNA was extracted from Caco-2 cells (90-95%, confluence) according to the manufacturer's instructions using Tri Reagent®-RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Inc., Cat. No: TR118). miR and mRNA were transcribed into cDNA with miRcute Plus miR First-Strand cDNA kit (TIANGEN, KR211) and HiScript®III 1st Strand cDNA Synthesis kit (+gDNA wiper; Vazyme, R312-02) respectively, according to the manufacturer's protocol. RT-qPCR analysis was performed using the miRcute Plus miRNA qPCR kit (SYBR Green; TIANGEN, FP411-02) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711). The thermocycling conditions were as follows: Pre-denaturation at 95˚C for 15 min, followed by 5 cycles of 94˚C for 20 sec, 60˚C for 30 sec and 72˚C for 34 sec, by 45 cycles of 94˚C for 20 sec and 60˚C for 34 sec. miR-146a-5p expression levels were normalized to U6. While, mRNA expression levels were normalized to GAPDH. Relative expression levels were calculated using the 2-ΔΔCq method (30 (link)). The oligonucleotide primers of target genes were designed and synthesized by TsingKe Biological Technology (Table I ).
5
Quantifying Viral RNA in Tears and Trachea
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Relative IBV RNA levels in tears and tracheas were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Viral RNA was extracted from individual tear samples using the QIAamp viral RNA mini kit (Qiagen, Valencia, CA), and from homogenized tracheas with TriReagent® RNA/DNA/protein isolation reagent (Molecular Research Center, Cincinnati, OH) following the manufacturers’ protocols. Relative viral RNA concentrations in tear and tracheal samples were determined by TaqMan® qRT-PCR as described [29] (link). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-test.
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using Tri-Reagent RNA/DNA/Protein Isolation Reagent (Molecular Research Center, Cincinnati, OH, USA) and then treated with RNase-free DNase (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was purified using an RNA Clean & Concentrator column (Zymo Research, Irvine, CA, USA). Two micrograms of DNase-treated total RNA was used to prepare cDNA by reverse transcription with M-MLV reverse transcriptase (New England Biolabs Inc., Ipswich, MA, USA) and oligo(dT)18 primers (Bioline, London, UK), according to the manufacturer’s protocol. Gene expression was determined by quantitative RT-PCR (MyiQ real-time PCR detection system, Bio-Rad Laboratories Inc., Hercules, CA, USA) using gene-specific primers (Table S1 ) and SensiMix SYBR & Fluorescein Kit (Bioline, London, UK), following the manufacturer’s instructions.
The Arabidopsis TON1A amplicon (At3g55000) was used as an internal control to normalize all data. The efficiency of the quantitative RT-PCR reactions was higher than 95%.
Quantitative RT-PCR reactions were carried out with cDNA synthesized from three pools of 36–60 roots, depending on the treatment used, harvested randomly.
The Arabidopsis TON1A amplicon (At3g55000) was used as an internal control to normalize all data. The efficiency of the quantitative RT-PCR reactions was higher than 95%.
Quantitative RT-PCR reactions were carried out with cDNA synthesized from three pools of 36–60 roots, depending on the treatment used, harvested randomly.
7
Investigating Testicular Gene Expression and Oxidative Stress
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After blood sample collection, the rats were euthanized and their abdomen was opened to get different tissue samples for different purposes:
Testicular tissue portion was used for RT-qPCR analysis of the studied genes (Cyp11a1, Nrf-2, Bax and Bcl-2). Thus, this portion was subjected to RNA isolation via TRI REAGENT® - RNA / DNA / PROTEIN ISOLATION REAGENT (Molecular Research Center, Inc. Cincinnati, OH., USA, Catalog number: TR 118) following the guidance of manufacturer’s protocol.
The second portion of testicular tissue was homogenized in phosphate buffer saline (PBS) to assess the oxidant-antioxidant markers.
The third portion of testis as well as specimens from epididymal, semen vesicular gland and prostatic tissue were all preserved in neutral buffered formalin (10%) for histological examination.
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