Total RNA was extracted from cultured cells and tissues using a Trizol standard protocol (Invitrogen, Carlsbad, USA). The integrity, quantity, and purity of RNA were examined using Nano-Drop 3000 Spectrophotometer (Thermo Scientific, Wilmington, USA). For each sample, 500 ng of total RNA was converted to cDNA using High Capacity cDNA Reverse Transcripition Kit (Applied Biosystems, Foster City, USA). The relative expression levels of mRNAs and miRNAs were quantified by the mirVana qRT-PCR miRNA Detection Kit in conjunction with real-time RT-PCR with SYBR Green I (Applied Biosystems). The threshold cycle (Ct) was determined and relative mRNA and miRNA levels were calculated based on the Ct values and normalized to GAPDH or U6 level for each sample. The sequences of primers used in our qRT-PCR experiments are shown in Table 1 .
Trizol standard protocol
Manufactured by Thermo Fisher Scientific
Sourced in United States
Trizol is a reagent used for the isolation and purification of RNA from biological samples. It is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components that effectively lyses cells and denatures proteins to release the RNA. The standard Trizol protocol involves sample homogenization, phase separation, RNA precipitation, and RNA washing steps to obtain high-quality, intact RNA.
Automatically generated - may contain errors
Lab products found in correlation
Mirvana mirna isolation kit, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent microarray scanner g2565ca, by Agilent Technologies (2 mentions)
Agilent scan control 7, by Agilent Technologies (2 mentions)
Labelit mirna labelling kit, by Mirus Bio (2 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Mirvana qrt pcr mirna detection kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 3000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcripition kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green 1, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Fast sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 fast, by Thermo Fisher Scientific (1 mentions)
Dnase 1 amp grade, by Thermo Fisher Scientific (1 mentions)
Real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green 1, by Toyobo (1 mentions)
High capacity cdna reverse transcription kit, by Toyobo (1 mentions)
10 protocols using trizol standard protocol
1
Quantifying mRNA and miRNA Expression
2
Quantitative Analysis of miR-93-3p and PEDF in ccRCC
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Total RNA was isolated from cultured cells or tissue sections (5 to 10 of 10 μm-thick) using a Trizol standard protocol (Invitrogen, USA). For formalin-fixed, paraffin-embedded (FFPE) ccRCC samples, total RNA was extracted from 5 to 10 of 10 μm-thick tissue sections using the Ambion RecoverAll kit (Ambion, USA) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate in the ABI 7500 fast real-time PCR System (Applied Biosystems, USA) and normalized with U6 and Actin endogenous control. Total RNA from Normal kidney tissues was used as a control. miR-93-3p, U6 levels and endogenous mRNA levels of PEDF and Actin were detected using SYBR Green PCR Master Mix kit in accordance with the manufacturer’s instructions (Applied Biosystems, USA). U6 was used as an internal control for miR-93-3p calculation and Actin were used as an internal control for detection of PEDF mRNA. The expression of miR-93-3p and PEDF mRNA were calculated using the comparative Ct method. Relative expression intensity values were calculated as 2-∆∆Ct.
3
Quantitative Analysis of mRNA and miRNA
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Total RNA was isolated from heart tissues or cultured cardiomyocytes using a TRIzol standard protocol (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was examined by NanoDrop 8000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Reverse transcription of RNA into cDNA used High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The relative expression levels of mRNAs and miRNAs were quantified by quantitative real-time PCR with SYBR Green I (Roche, Indianapolis, IN, USA). After circle reaction, the threshold cycle (Ct) was determined, and relative mRNA and miRNA levels were calculated based on the Ct values and normalized to β-actin or U6 level in each sample. The specific primers used in this study were shown as follows: U6 RT primer, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; miR-26a RT primer, 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCTAT-3′; U6 forward, 5′-CGCTTCACGAATTTGCGTGTCAT-3′ and reverse, 5′-GCTTCGGCACATATACTAAAAT-3′; miR-26a forward, 5′-GCGTAGCAGCGGGAACAGT-3′ and reverse, 5′-CCAGTGCGTGTCGTGGAGT-3′; MIRF forward, 5′-TCTTTCCCAGTTCTCCTTGG-3′ and reverse, 5′-GCAGTAGCAAATTCCCCAAA-3′; Bak1 forward, 5′-CAGGATGGGGTCTCTACGAA-3′ and reverse, 5′-GGGCTTTGGCTACCGTCT-3′.
4
Comprehensive miRNA Profiling of CNS Tumors
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MicroRNA profiling has been described previously in detail [3 (link),4 (link),5 (link)], which we reproduce in quotes as follows: “In brief, total RNA and miRNAs were extracted using the Trizol standard protocol (Invitrogen, Carlsbad, CA, USA) and the mirVANA miRNA isolation kit (Ambion, Austin, TX, USA). Labelling and hybridization were performed using the LabelIT miRNA labelling kit (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s instructions. Samples were hybridized to Applied MicroArrays (miRlink Bioarray 300054-3PK) platform. This array contained 1211 human miRNAs. Hybridization was performed at 37 °C with rotation at 145 rpm for 16 h. Images were scanned using Agilent Microarray Scanner (G2565CA) controlled by Agilent Scan Control 7.0 software. The total gene signals were extracted using the Imagene 6.0 software (Biodiscovery Inc., El Segundo, CA, USA) that contains summarized signal intensities for each miRNA by combining intensities of replicate probes and background subtraction” [3 (link),4 (link),5 (link)]. In total, 49 CNS tumor samples (the complete cohort described in Section 2.1 .) and 13 control samples were investigated for their miRNA expressional profile. All microarray data are MIAME compliant.
5
Total RNA and miRNA Extraction from Samples
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Samples (n = 61) were processed for both total RNA, as well as miRNA extraction. In brief, total RNA and miRNAs were extracted using the Trizol standard protocol (Invitrogen, Carlsbad, CA, USA) and the mirVANA miRNA isolation kit (Ambion, Austin, TX, USA). The RNA quantity and quality were evaluated using a spectrophotometer (NanoDrop® ND-1000 UV–vis, Nanogen Inc., San Diego, CA, USA), as previously described [3 (link),4 (link),5 (link)].
6
Quantitative Gene Expression Analysis
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Total RNA was isolated from samples using TRIzol standard protocol (Invitrogen, CA, USA), according to manufacturer’s instruction. First-strand cDNA synthesis was performed using RevertAid RT Reverse Transcription Kit (Thermo Fisher, USA). The expression levels of c-Myc and Rab7a were analyzed using Fast SYBR Green PCR Master Mix (Applied Biosystems, USA). β2-MG was used as the internal control for both c-Myc and Rab7a. The qRT-PCR was performed on thermocycler ABI Prism 7500 fast (Applied Biosystems, CA, USA). All of the data were normalized to the internal control. Forward and reverse primers for each gene are listed below c-Myc: 5'-CCCTCAGTGGTCTTTCCCTAC-3', 5'-CTTGTCGTTTTCCTCCGTGT-3'; Rab7a: 5'-ACCAGTACAAAGCCACAATAGG-3', 5'-GGGGCAGTCACATCAAACAC-3'; β2-MG: 5'-CTATCCAGCGTACTCCAAAGAT-3', 5'-ACACGGCAGGCATACTCATC-3'.
7
Comprehensive miRNA Profiling Workflow
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In brief, total RNA and miRNAs were extracted using the Trizol standard protocol (Invitrogen, Carlsbad, CA) and the mirVANA miRNA isolation kit (Ambion, Austin, TX). The RNA quantity and quality were evaluated using a spectrophotometer (NanoDrop® ND-1000 UV–vis, Nanogen Inc.). Labelling and hybridization were performed using the LabelIT miRNA labelling kit (Mirus Bio LLC, USA) according to manufacturer’s instructions. Samples were hybridized to Applied MicroArrays (miRlink Bioarray 300054-3PK) platform. This array contained 1211 human miRNAs. Hybridization was performed at 37°C with rotation at 145 rpm for 16 h. Images were scanned using Agilent Microarray Scanner (G2565CA) controlled by Agilent Scan Control 7.0 software.
8
Liver RNA Extraction and Purification
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Total liver RNA was isolated using the TRIZOL standard protocol (Invitrogen Life Technologies, Milano, Italy). Tissue/TRIZOL mixtures were homogenized using a polytron, keeping the viscosity of the solution to a minimum to ensure effective inactivation of endogenous RNAse activity. First-strand RNA was treated with DNase I, Amp Grade, to remove residual genomic DNA (Invitrogen Life Technologies).
9
Real-Time PCR-Based Quantification of lncRNA and miRNA
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Total RNA was extracted from cultured cells using a Trizol standard protocol (Invitrogen, Carlsbad, United States). The quantity and purity of RNA were examined using Nano-Drop 8,000 Spectrophotometer (Thermo Scientific, Wilmington, United Staates). For each sample, 500 ng total RNA was reverse transcribed into cDNA with High Capacity cDNA Reverse Transcription Kit (TOYOBO, Osaka, Japan), and then amplified with SYBR Green I (TOYOBO, Osaka, Japan) using 7,500 Real Time-PCR System (Applied Biosystems, Foster City, CA, United States). The threshold cycle (Ct) was determined. Relative lncRNA and miRNA levels were calculated based on the Ct values and normalized to GAPDH or U6 level for each sample. The sequences of primers are shown in Table 1 .
10
Quantitative Analysis of miR-195 and CX3CL1
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Total RNA was isolated from specimens and cells using a Trizol standard protocol (Invitrogen) and extracted from plasma using the miRNeasy Serum/Plasma Kit (QIA-GEN). Quantitative RT-PCR was performed in triplicate using the ABI 7900 fast real-time PCR System (Applied Biosystems) and normalized with U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) endogenous control. MiR-195 and U6 levels were measured using the Taq-Man microRNA assay kit, and mRNA levels of CX3CL1, CX3CR1, and GAPDH were detected using the SYBR Green PCR Master Mix kit in accordance with the manufacturer's instructions (Applied Biosystems).
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