RNA from EXOs was isolated using miRNeasy columns (Qiagen) according to the manufacturer’s protocol; total RNA from cell lines was extracted with a TRIzol extraction kit (Life Technologies, Grand Island, NY, USA). Nucleic acid quality and quantity were assessed with a NanoDrop spectrophotometer (NanoDrop Technologies, ThermoFisher Scientific). Total RNA for each sample was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s protocols. qPCR was performed on a ViiA7 system (Life Technologies) using TaqMan PCR Master Mix (Life Technologies). Predesigned TaqMan probes (Life Technologies) were used for Matrix Metalloproteinase 1 (MMP1, Hs00899658_m1), Matrix Metalloproteinase 9 (MMP9, Hs00957562_m1), Cyclin D1 (CCND1, Hs00765533_m1), CD99 (Hs00908458) and miR-199a-3p (Hs002304). Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).
Trizol extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The TRIzol extraction kit is a reagent designed for the isolation and purification of total RNA from various biological samples. It utilizes a monophasic solution of phenol and guanidine isothiocyanate to facilitate the separation of RNA from DNA, proteins, and other cellular components.
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Lab products found in correlation
M mlv reverse transcriptase, by Thermo Fisher Scientific (11 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Primescript rt pcr kit, by Takara Bio (2 mentions)
Lipofectamine 2000 transfection kit, by Thermo Fisher Scientific (2 mentions)
Mtt kit, by Beyotime (2 mentions)
Facscanto flow cytometer, by BD (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
7300 rt pcr system, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Amv reverse transcriptase, by Promega (2 mentions)
Rnase free dnase 1, by Takara Bio (2 mentions)
Cfx manager system, by Bio-Rad (2 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Taqman predesigned probes, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
78 protocols using trizol extraction kit
1
Quantitative Analysis of Exosomal RNA
2
Quantification of Chemokine Expression in Mouse Lungs
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Mouse lung tissues were collected. Total RNA was extracted by using the TRIzol extraction kit (Life Technologies). Expression levels of chemokines (CXCL1 and CXCL2) were determined by quantitative real-time RT-PCR with a Bio-Rad thermocycler and an SYBR green kit (Bio-Rad) according to the recommendations of the manufacturer. Mouse Gapdh was used as reference gene. The primers had the following sequences: CXCL1 forward 5′-TCGATGGTAGTTCAGTTCTGCT-3′ and reverse 5′-TCGCACAACACCCTTCTACT-3′, CXCL2 forward 5′-CCCTGCCAAGGGTTGACTTC-3′ and reverse-5′- GGGGCTTCAGGGTCAAGG-3′, Gapdh forward 5′-GCGAGATCCCGCTAACATCA-3′, and reverse 5′-CTCGTGGTTCACACCCATCA-3′. All samples were run in triplicate. Relative levels of mRNA were normalized to the mouse housekeeping gene, and the results are expressed as fold change vs. the sham controls.
3
Total RNA Extraction Using Trizol
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Total RNA was extracted from cells using Trizol extraction kit (Life Technologies, 15596018) according to the manufacturer's protocol. The quantity and purity of RNA was determined by OD260/280 reading using a Nanodrop spectrophotometer.
4
Quantitative Analysis of Stem Cell Markers
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RNA extraction was carried out using the Trizol extraction kit according to manufacturer' s instruction (Life Technologies-China, Shanghai, China), with purified RNA samples stored at −70°C until use. cDNAs were produced by retrograde transcription using commercial kits including Accupower, RocketScript and RT PreMix (Bioneer-China, Shanghai, China). Specific DNA fragments were amplified using RealMasterMix (Probe) and miRcute miRNA with a Stratagene cycler (Tiangen Biomart, Beijing, China). The following forward (F) and reverse (R) primers were used for qPCR amplifications (synthesized by Bioneer-China). LGR5-F: 5′-TGCCATTATTCACCCCAAC-3′ ; LGR5-R: 5′-CACAGCACTGGTAAGCGTATG-3′ ; ephrin-B3-F: 5′-GCTGCTGTTAGGTTTTGCG-3′ ; ephrin-B3-R: 5′-CCCCGATCTGAGGGTAAAG-3′ ; Msi-1-F: 5′-AGGGGTTTCGGCTTCGT-3′ ; Msi-1-R: 5′-TGACCATCTTAGGCTGTGCTC-3′ ; PCNA-F:5′-GGTGAAGTTTTCTGCGAGTG-3′; PCNA-F: 5′-GGTGAAGTTTTCTGCGAGTG-3′ ; rat β-actin-F: 5′-GTCAGGTCATCACTATCGGCAAT-3′ ; and rat β-actin-R: 5′-AGAGGTCTTTACGGATGTCAACGT-3′ .
5
Osteogenic Differentiation Protocol
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Four days after seeding cells were exposed to specific osteogenic medium, IMDM supplemented with 2 % FBS, 5 mM β-glycerophosphate and 50 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO) and maintained in differentiative conditions up to 14 days. Medium was renewed every 4 days. Cultures were harvested at various time points to collect RNA. Total RNA was extracted by the TRIzol extraction kit (Life Technologies, Grand Island, NY). Quality and quantity of RNA samples were assessed with NanoDrop analysis (NanoDrop Technologies). Expression of CD99 was verified by real-time PCR at basal and differentiative conditions in all profiled samples (Additional file 11 : Figure S6).
6
MCF7/AC1 Cell Transcriptome Sequencing
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RNA was prepared from MCF7/AC1 cells using the TRIzol extraction kit (Life Technologies). Genomic DNA was removed using the Ambion DNA-free kit. NuGEN Encore reagents were used for library preparation of total RNA samples. One microgram of total RNA input was used for each sample. The libraries were sequenced on an Illumina HiSeq 2000 sequencing system using 100-bp single-ended reads. After removing the poor-quality bases from FASTQ files for the whole transcriptome sequencing, paired-end reads were aligned by reads that were aligned to the human reference genome UCSC hg19 with Tophat 2.0.14 and the bowtie 2.2.6 aligner option. Transcript abundance was estimated using a count-based method with htseq-count.
7
Quantification of miRNA and mRNA
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Total RNA was isolated using a Trizol extraction kit (Life Technologies, USA) according to the manufacturer’s instructions. Purified mRNA and miRNAs were detected by qRT-PCR assay using All-in-One miRNA qRT-PCR Detection Kit (GeneCopoeia, USA). U6 small RNA was used as an internal control for normalization and quantification of miR-320b expression. As an internal control β-actin was measured for normalization and quantification of c-Myc expression.
8
Quantifying LINC00460 and miR-320b Expression
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The separation of total RNA was isolated from SW1990 cells was done with a Trizol extraction kit (Life Technologies, USA) following the manufacturer’s protocol. And the reverse-transcription of it into complementary DNA (cDNA) was done with a Prime-Script RT reagent kit (Takara Bio, Inc., Otsu, Japan). Purified mRNA and miRNAs were detected by qRT-PCR assay in combination with SYBR-green real-time Master Mix (Toyobo, Co., Ltd, Osaka, Japan) on an ABI Prism 7300 Sequence Detection system. As an internal control, GAPDH and U6 were measured for normalization and quantification of the expressions of LINC00460 and ARF1, or miR-320b expression, respectively. The 2−ΔΔCt method was applied to calculate the relative expression of target gene.
9
Xiangsu Pig Tissue RNA Extraction
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DNA was extracted from 184 blood samples of Xiangsu pigs using the whole blood DNA extraction kit (D3392-01, Omega). Total RNA of heart, liver, spleen, lung and kidney of Xiangsu pigs at 6 months of age and total RNA of longissimus dorsi muscle at newborn, 6 months and 12 months of age was extracted using the TRIzol Extraction Kit (15,596,026; Thermo Fisher). The concentration and purity of DNA and RNA were determined using an ultra-micro spectrophotometer (Thermo Mano Drop 2000), followed DNA by storage at −20°C and RNA by storage at −80°C (Supplementary Table S1 ).
10
Quantifying Apoptosis-Related Genes
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Real-time fluorescence quantitative PCR was used to detect the relative expression of the Bax and caspase-3 genes in each group of cells. According to the trizol extraction kit (15596026; Thermo Fisher Scientific, Waltham, MA, USA) experimental instructions, total RNA was extracted from the cells, and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific). An Invitrogen reverse transcription kit (K1691; Thermo Fisher) was used for reverse transcription. Different primers were designed based on the Bax and the caspase-3 genes. The primer sequences are shown in Table 1 and were synthesized by Biotechnology Co., Ltd. (Shanghai, China). PCR using 2 × Power Taq PCR mastermix (PR1702; Biotek, Beijing, China) and SYBR Green I (SY1020; Solarbio, Beijing, China) was performed on a fixed light quantifier. The relative expression of these two genes was calculated by the formula 2−ΔΔCT.
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