Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA). Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). Quantitative RT-PCR was performed according to a standard protocol using gene-specific primers (Supplementary Table 1 ). SYBR Green reactions were done using FastMix, Low ROX (Quanta Biosciences, Gaithersburg, MD) and products measured on an ABI Viia 7 PCR system (ABI, Foster City, CA). The expression of individual genes was calculated and normalized with the ΔΔCt method.
Trizol reagent protocol
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other components designed for the isolation of total RNA from various biological samples. The reagent maintains the integrity of RNA while disrupting cells and dissolving cell components.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Iq sybr green supermix, by Bio-Rad (4 mentions)
Superscript 3 first strand synthesis supermix kit, by Thermo Fisher Scientific (4 mentions)
Amplification grade dnase 1, by Merck Group (3 mentions)
Light cycler thermal cycler system, by Roche (3 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (3 mentions)
Nd 1000, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Rq1 rnase free dnase, by Promega (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
C1000 touch thermal cycler, by Bio-Rad (3 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (3 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
79 protocols using trizol reagent protocol
1
Quantitative RT-PCR for Lung RNA Analysis
2
Tambaqui Liver RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of total RNA from four tambaqui liver from each treatment group followed
the TRIzol® reagent protocol (Invitrogen) according to the manufacturer’s
instructions. Contaminating genomic DNA was removed using DNase I (Invitrogen).
First strand cDNA was reverse-transcribed from the total RNA using RevertAid H Minus
First Strand cDNA Synthesis kit (Fermentas), and following the manufacturer’s
instructions. Enzymatic treatment with reverse transcriptase (MMLV Reverse
Transcriptase) (200 U/μL, USB) was first done, and then mixed in a 0.2 mL microtube
with approximately 25 μg RNA, 1 μL of Oligo(dT)18 primer (1 μg), 1.0 μL
dNTP mix (10 mM), MMLV buffer 5X, and deionized water for a 20 μL final volume. This
solution was incubated at 37 °C for 1 h for conversion and at 70 °C for 10 min to
inactivate the enzyme.
the TRIzol® reagent protocol (Invitrogen) according to the manufacturer’s
instructions. Contaminating genomic DNA was removed using DNase I (Invitrogen).
First strand cDNA was reverse-transcribed from the total RNA using RevertAid H Minus
First Strand cDNA Synthesis kit (Fermentas), and following the manufacturer’s
instructions. Enzymatic treatment with reverse transcriptase (MMLV Reverse
Transcriptase) (200 U/μL, USB) was first done, and then mixed in a 0.2 mL microtube
with approximately 25 μg RNA, 1 μL of Oligo(dT)18 primer (1 μg), 1.0 μL
dNTP mix (10 mM), MMLV buffer 5X, and deionized water for a 20 μL final volume. This
solution was incubated at 37 °C for 1 h for conversion and at 70 °C for 10 min to
inactivate the enzyme.
3
Embryonic RNA-Seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from pooled samples of 8–10 embryos with four pools for each exposure using the TRIzol reagent protocol (Invitrogen, Carlsbad, CA, USA). Briefly, TRIzol was added to frozen embryos, before allowing embryos to thaw. Samples were homogenized in a Bead Mill 4 (Fisher Scientific, PA) using sterile 2 mm glass beads and resuspended in 50 µL RNase-free water. The quantity and quality of samples were determined using a NanoDrop 2000 (A260/280 and A260/230 > 1.8). Between 500 and 750 ng of total RNA per sample were delivered to the Scripps Research Genomics Core (San Diego, CA, USA) for library prep and shallow RNA sequencing (RNA-Seq).
Sample quality was assessed using a 2100 Bioanalyzer 2100 (Agilent Technologies) (RIN > 8.5). Library preparation was performed using the HTP RNA-Seq Library Prep Kit (iGenomX Inc, San Francisco, CA, USA). Briefly, barcoded oligo dT primers were added to 50 ng RNA per sample and reverse transcribed. Sample cDNA products were combined, cleaned, and run through PCR with 0.5 µM barcoded PCR primers (p5 and p7 sequences, Illumina, San Diego, CA, USA). Purified PCR products were sequenced using a NextSeq2000 sequencer (paired-end mode; read1: 26 bp, read2: 94 bp), generating greater than 1.5 million paired-end reads for each sample.
Sample quality was assessed using a 2100 Bioanalyzer 2100 (Agilent Technologies) (RIN > 8.5). Library preparation was performed using the HTP RNA-Seq Library Prep Kit (iGenomX Inc, San Francisco, CA, USA). Briefly, barcoded oligo dT primers were added to 50 ng RNA per sample and reverse transcribed. Sample cDNA products were combined, cleaned, and run through PCR with 0.5 µM barcoded PCR primers (p5 and p7 sequences, Illumina, San Diego, CA, USA). Purified PCR products were sequenced using a NextSeq2000 sequencer (paired-end mode; read1: 26 bp, read2: 94 bp), generating greater than 1.5 million paired-end reads for each sample.
4
Transcriptomic Analysis of Soybean-Sclerotinia Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from soybean stem tissues and infecting S. sclerotiorum using a modified TRIzol™ Reagent protocol (Invitrogen Corp., Carlsbad, CA, USA). Further, samples were cleaned using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA concentration and purity was determined by Nanodrop (Thermo Fisher Scientific, Wilmington, DE) and sample quality was assessed using an Agilent Bioanalyzer 2100 and an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). Three biological replicates were generated per treatment.
Library preparation was performed at the University of Wisconsin – Madison Biotechnology Centre (Madison, WI, USA). Individually indexed libraries were prepared using the TruSeq RNA Sample Preparation v2 kit according to the manufacturer’s instructions (Illumina, San Diego CA, USA). Library concentrations were quantified with the Qubit HS DNA kit (Thermo Fisher Scientific, Wilmington, DE). The size and quality of the libraries were evaluated with an Agilent Bioanalyzer 2100 and an Agilent DNA 1000 kit (Agilent Technologies, Santa Clara, CA) and the libraries were sequenced using Illumina HiSeq2500 (1X100bp) (Illumina, San Diego CA, USA).
Library preparation was performed at the University of Wisconsin – Madison Biotechnology Centre (Madison, WI, USA). Individually indexed libraries were prepared using the TruSeq RNA Sample Preparation v2 kit according to the manufacturer’s instructions (Illumina, San Diego CA, USA). Library concentrations were quantified with the Qubit HS DNA kit (Thermo Fisher Scientific, Wilmington, DE). The size and quality of the libraries were evaluated with an Agilent Bioanalyzer 2100 and an Agilent DNA 1000 kit (Agilent Technologies, Santa Clara, CA) and the libraries were sequenced using Illumina HiSeq2500 (1X100bp) (Illumina, San Diego CA, USA).
5
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene expression levels were quantitated by the method of real-time PCR. Firstly, RNA from the frozen samples were isolated using the Trizol Reagent protocol (Invitrogen, Carlsbad, CA). Then cDNA was synthesized using a PrimeScript® RT reagent kit (Takara, Dalian, China) with gDNA Eraser. Finally, the real-time PCR was carried out with the SYBR® Premix Ex Taq™ (Takara, Dalian, China) on 7,500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Ribosomal protein L4 (RPL4) was used as the reference gene. Data was analyzed by using the 2−ΔΔCt method as described previously (17 (link)). The primer sequences are shown in Table 2 .
6
Rumen Microbiome RNA Isolation and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The frozen rumen samples were ground using liquid nitrogen. About 0.5 g of frozen fine powder was used for total RNA isolation using Trizol-Reagent protocol (Invitrogen, Carlsbad, CA, USA), followed by RNA clean up using MEGA clear Kit (Invitrogen, Carlsbad, CA, USA). Total RNA quality and quantity were estimated using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA 6000 Nano kit (Agilent Technologiess, Santa Clara, CA, USA, USA). One hundred nanogram of total RNA was reverse-transcribed into first strand cDNA and sequenced using Illumina rRNA MiSeq preparation kit (Illumina, San Diego, CA, USA) by Illumina MiSeq platform.
7
Kidney mRNA Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney tissue was disrupted using liquid nitrogen and grinded thoroughly with a mortar. Total RNA was isolated using TRIzol reagent protocol (Invitrogen, Carlsbad, CA, USA), and a treatment with DNAse I amplification Grade (Sigma-Aldrich, St. Louis, MO, USA) was done according to the manufacturer’s instruction. Quantification was performed by spectrophotometry (ND1000, Nanodrop Technologies, Wilmington, DE, USA). Fifty nanograms of total RNA were used to analyze mRNA expression in the Light Cycler thermal cycler system (Roche Diagnostics, Indianapolis, IN, USA). QuantiTect SYBR Green RT-PCR kit (Qiagen GmbH, Hilden, Germany) was used for quantification following the manufacturer’s protocol. Results were normalized to GAPDH by using the 2−ΔΔCt method. Primers for CYP27b1 and CYP24a1 quantification were purchased from Sigma Aldrich (Sigma-Aldrich St. Louis, MO, USA). Sequences for GAPDH were purchased from Eurofins (Eurofins Genomics, Germany GmbH, Ebersberg, Germany) (Table 2 ).
8
Quantifying Drosophila Transcript Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from brains and ventral nerve ganglia of 40 third instar larvae following the standard Trizol reagent protocol (Invitrogen). Power SYBR Green PCR Master Mix (Applied Biosystems) was used for quantitative real-time PCR. Actin mRNA level was amplified as an internal control using published primers [33 (link)], whereas tkv cDNA was amplified with primers 5’-GTG ATA GGG CAG GGC GTA GT-3’ and 5’-AGT GGG TCT CGT TCT GTG GG-3’.
9
Quantifying FRα mRNA Expression in Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR was used to measure the mRNa expression of FRα in the 3 cancer cell lines. Total RNA was extracted from the 3 types of cancer cells using TRIzol reagent protocol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using 500 ng of total RNA and the PrimeScript™ RT reagent kit according to the manufacturer's instructions, and PCR was performed using the following primers: human FRα (sense strand, 5′-AGGTGCCATCTCTCCACAGT-3′ and antisense, 5′-GAGGACAAGTTGCATGAGCA-3′; cDNA amplicon size: 135 bp; Tm, 60°C); human GAPDH (sense, 5′-TTAAAAGCAGCCCTGGTGAC-3′ and antisense, 5′-CTCTGCTCCTCCTGTTCGAC-3′; cDNA amplicon size: 138 bp; Tm, 60°C). cDNA was measured by using SYBR-Green RT-PCR in a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 95°C for 30 sec, 40 cycles of 95°C for 5 sec and 60°C for 34 sec. All PCR reactions were performed at least in triplicate and the mRNA levels were represented as relative quantification, which was calculated using relative expression of FR (NFRα) = 2−ΔΔCq, where Cq = ΔCq sample − ΔCq calibrator (ΔCq = Cq of target gene − Cq of GAPDH).
10
RNA Extraction and cDNA Synthesis Protocol
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!