Transcriptome sequencing was conducted on muscle samples obtained 40 days after SsMeox1 knockdown with 3 knockdown samples and 3 control samples. RNA extraction, sequencing library construction, and Illumina sequencing were performed by Novogene Bioinformatics Technology Co., Ltd. Briefly, the total RNA of the six samples (three biological replications per group) was extracted using Invitrogen TRIzol (ThermoFisher, Waltham, MA, USA). The RNA concentration and quality were analyzed using a Fragment Analyzer 5400 (Agilent, Santa Clara, CA, USA). The NEBNext® UltraTM RNA Library Prep Kit (NEB, Ipswich, MA, USA) was utilized for library preparation. The library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). These libraries were sequenced using the NovaSeq 6000 platform. Salmon version 0.7.2 was used for the quantification of the transcript counts [48 (link)]. The gene expression levels were standardized by the transcripts per kilobase million (TPM) [49 (link)]. The analysis of differentially expressed genes (DEGs) was performed using the R package DESeq2 [50 (link)], and the genes with |log2 FC| ≥ 1 and an adjusted p-value < 0.01 were assigned as DEGs. In addition, GO and KEGG enrichment analysis were performed by DAVID (https://david.ncifcrf.gov/ (accessed on 14 October 2021)), and the results were visualized by R Studio.
Invitrogen trizol
Manufactured by Thermo Fisher Scientific
Sourced in United States
Invitrogen TRIzol is a reagent used for the isolation of total RNA from a variety of biological samples, including cells, tissues, and body fluids. It is based on the single-step RNA isolation method developed by Chomczynski and Sacchi.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Fragment analyzer 5400, by Agilent Technologies (1 mentions)
Nebnext ultratm rna library prep kit, by New England Biolabs (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Quantstudio system, by Thermo Fisher Scientific (1 mentions)
Mirna first strand cdna synthesis tailing reaction, by Sangon (1 mentions)
M6a rna methylation quantification kit, by Epigentek (1 mentions)
Nanodrop technology, by Thermo Fisher Scientific (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Stepone plus real time pcr thermocycler, by Thermo Fisher Scientific (1 mentions)
Sybr green real time pcr master mix, by Thermo Fisher Scientific (1 mentions)
Reverse transcription, by Transgene (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy spin column, by Qiagen (1 mentions)
Rna nano chip kit, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
15 protocols using invitrogen trizol
1
Transcriptomic Analysis of SsMeox1 Knockdown
2
Comprehensive Transcriptome Analysis Protocol
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Total RNA was extracted from cell cultures and tissues with Invitrogen TRIzol (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. For miRNA, we used miRNA First Strand cDNA Synthesis (Tailing Reaction) (Sangon Biotech, Shanghai, China), and the miRNA levels were normalized to the uniformly expressed U6 snRNA. For mRNA, we employed RevertAid First Strand cDNA Synthesis Kit and PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, USA), and assessed the mRNA expression by qRT-PCR on a QuantStudio System (Thermo Fisher Scientific, USA). All primers used are listed in Supplementary Table S3 . Threshold cycles (Cts) were generated automatically, and the relative expressions were present as 2−ΔCt. The mRNA levels were normalized to ACTB.
3
Quantification of m6A RNA Methylation
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Total RNA from patient samples was isolated using Invitrogen TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, and treated with deoxyribonuclease I (Sigma-Aldrich; Merck KGaA, USA). RNA quality was analyzed using NanoDrop technology (Thermo Fisher Scientific, USA). The m6A RNA methylation quantification kit (Epigentek, USA) was used to measure the m6A content in the total RNAs. Briefly, 200 ng RNAs were coated on assay wells. Capture antibody solution and detection antibody solution were then added to assay wells separately, following the manufacturer’s instructions. The m6A levels were quantified colorimetrically by reading the absorbance of each well at a wavelength of 450 nm, and then calculations were performed based on the standard curve.
4
Quantitative RT-PCR Analysis of mRNA
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The RNA was extracted from cells with Invitrogen TriZol (Thermo Fisher Scientific, Waltham, MA, USA). Out of the extract, 1 µg was reverse-transcribed into cDNA (Qiagen, Hilden, Germany) and amplified with SYBR Green Real-Time PCR Master Mix in a StepOne Plus real-time PCR thermocycler (Applied Biosystems, Carlsbad, CA, USA). The sequence of primers is listed in detail in Supplementary Table S3 . The PCR results for each type of mRNA were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase and expressed as fold change in the Ct value relative to the control group in accordance with the 2–ΔΔCt method.11 (link)
5
Quantifying Gene Expression by Real-Time PCR
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Total RNA was extracted with Invitrogen Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using a UV spectrophotometer. After reverse transcription (Transgen, Beijing, China), RCR amplification was performed by Real-Time PCR using the Fermentas SYBR Green PCR reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, in an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific). Results were processed using the ABI 7300 SDS software (Thermo Fisher Scientific) and normalized to GAPDH mRNA levels. Each reaction was performed in triplicate.
6
RNA Extraction from Ovarian Tumor Samples
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Tumors from patients with ovarian cancer (serous and endometrioid adenocarcinoma) were collected and stored at –80°C for later use. Tumors weighing 30–40 mg were placed in 750 µL Invitrogen TRIzol (Thermo Fisher Scientific) and cut into smaller pieces with scissors. To each sample 150 µL of chloroform was added, and samples were mixed for 30 seconds and centrifuged at 20,854 × g for 15 minutes at 4°C to separate RNA into the aqueous phase. The aqueous phase was mixed with approximately 0.53 × volume of 100% ethanol and transferred to an RNeasy spin column (Qiagen). Total RNA was purified with RNeasy Mini kit (Qiagen) as per manufacturer's protocol with RNAse free DNase (Qiagen) on-column treatment. RNA concentration and integrity were measured on the 2100 Bioanalyzer (Agilent Technologies) using the RNA NanoChip Kit (Agilent).
7
Quantitative Analysis of Antioxidant Pathways
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The total RNA was isolated and extracted from treated spermatozoa groups using
the Invitrogen TRIzol (Thermo Fisher Scientific). One microgram of total RNA was
converted into complementary DNA using random hexamers and M-MLV (Thermo Fisher
Scientific). The quantitative reverse transcription polymerase chain reaction
(qRT-PCR) was performed in iCycler Multicolor RT-PCR system using SYBR Green
(Bio-Rad, Hercules, California). The expression of tested gene was normalized to
internal control GAPDH, and fold change was calculated by the
2−ΔΔCT method. Primers for the qRT-PCR were listed below:
Nrf2, upstream 5′-ACAGTGCTCCTATGCGTGAA-3′, downstream
5′-GAGCCTCTAAGCGGCTTGAA-3′; HO-1, upstream 5′-CAGAAGAGGCTAAGACCGCC-3′,
downstream 5′-CTCTGACGAAGTGACGCCAT-3′; and GAPDH (internal reference), upstream
5′-ACCACAGTCCATGCCATCAC-3′ and downstream 5′-TCCACCACCCTGTTGCTGTA-3′. In all of
these qRT-PCR measurements, assays of samples were performed in duplicate. Three
or more independent experiments were carried out and assayed.
the Invitrogen TRIzol (Thermo Fisher Scientific). One microgram of total RNA was
converted into complementary DNA using random hexamers and M-MLV (Thermo Fisher
Scientific). The quantitative reverse transcription polymerase chain reaction
(qRT-PCR) was performed in iCycler Multicolor RT-PCR system using SYBR Green
(Bio-Rad, Hercules, California). The expression of tested gene was normalized to
internal control GAPDH, and fold change was calculated by the
2−ΔΔCT method. Primers for the qRT-PCR were listed below:
Nrf2, upstream 5′-ACAGTGCTCCTATGCGTGAA-3′, downstream
5′-GAGCCTCTAAGCGGCTTGAA-3′; HO-1, upstream 5′-CAGAAGAGGCTAAGACCGCC-3′,
downstream 5′-CTCTGACGAAGTGACGCCAT-3′; and GAPDH (internal reference), upstream
5′-ACCACAGTCCATGCCATCAC-3′ and downstream 5′-TCCACCACCCTGTTGCTGTA-3′. In all of
these qRT-PCR measurements, assays of samples were performed in duplicate. Three
or more independent experiments were carried out and assayed.
8
Profiling TGFβ2 Response in Stem Cells
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AAP and SC cells were cultured in six-well plates with duplicates from each batch or each strain. After reaching 80% confluence, three batches of primary bovine AAP cells or three strains of primary human SC cells were treated with or without recombinant human transforming growth factor beta-2 (TGFβ2) protein (Abcam) at 5 ng/mL and 10 ng/mL in fetal bovine serum-free culture medium for 24 or 48 hours. Total RNA was then isolated using Invitrogen TRIzol (Thermo Fisher Scientific) according to the manufacturer's recommended protocol for further gene expression profiling.31 For consistent expression profiling, we examined the gene expression response of TGFβ2 treatment in SC cells using Bio-Rad ddPCR assays as previously described.30 (link),32 –37 (link) All of the ddPCR assays are listed in Table 2 .
9
Genomic Diversity of Rare Pheasant Species
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All procedures used for this study that involved birds complied with guidelines for the care and utility of experimental animals established by the Ministry of Agriculture of China and the Jiangxi Forestry Bureau (permission number: 2006-398). The ethics committee of Jiangxi Science & Technology Normal University approved this study. The samples used in this study were collected from different areas, among which GCO was a captive population raised in the Nanchang Zoo with permission from relevant authorities. The 14 GCO individuals were captured at different locations in Wuyuan during 2014–2016 for the purpose of ex situ conservation. We collected blood samples from the wing veins of 66 birds, including 14 GCO, six GCA, six GCH, five GPE, 11 GSA, eight GBE, seven GCI, eight TMI, and one outgroup of azure-winged magpie (Cyanopica cyana, CCY). Genomic DNA was extracted using a Whole Blood DNA Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA integrity was verified with agarose gel electrophoresis. Two adult females of GCO and GSA were selected for de novo assembly. The liver and muscle tissues were collected from individuals suffering accidental death and mixed to extract RNA using Invitrogen TRIzol (Thermo Fisher Scientific) for subsequent gene functions annotation.
10
Cell Culture and Characterization of Bone Cells
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Cells were cultured with α-modified essential medium (α-MEM; Hyclone, South Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA) in a 5% CO2 and 95% air incubator at 37 °C. Preosteoclast-like Raw264.7 cells and preosteoblast-like MC3T3-E1 cells were purchased from ATCC (ATCC, Manassas, VA, USA). Osteocyte-like MLO-Y4 cells were received as a gift from Prof. Lynda F. Bonewald35 (link).
Fluorescent dyes DIL, Hoechst and DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant mouse receptor activator of NF-ƙB ligand (RANKL), parathyroid hormone (PTH) and macrophage colony-stimulating factor (M-CSF) were obtained from Peprotech (Rocky Hill, NJ, USA). 1,25(OH)2D3 was acquired from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA). Tartrate-resistant acid phosphatase reagents were purchased from Sigma (TRAP, Aldrich Sigma, St. Louis, MO, USA). RNA sample preparation was carried out according to the standard protocols of Invitrogen™ TRIzol™ (Thermo Fisher Scientific, Waltham, MA USA). Phosphate buffer saline (PBS), trypsin and 4% paraformaldehyde solution (4%PFA) were purchased from Boster China (Boster, Wuhan, China).
Fluorescent dyes DIL, Hoechst and DAPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant mouse receptor activator of NF-ƙB ligand (RANKL), parathyroid hormone (PTH) and macrophage colony-stimulating factor (M-CSF) were obtained from Peprotech (Rocky Hill, NJ, USA). 1,25(OH)2D3 was acquired from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA). Tartrate-resistant acid phosphatase reagents were purchased from Sigma (TRAP, Aldrich Sigma, St. Louis, MO, USA). RNA sample preparation was carried out according to the standard protocols of Invitrogen™ TRIzol™ (Thermo Fisher Scientific, Waltham, MA USA). Phosphate buffer saline (PBS), trypsin and 4% paraformaldehyde solution (4%PFA) were purchased from Boster China (Boster, Wuhan, China).
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