Frozen placental samples were ground into a powder using liquid nitrogen-cooled mortar and pestle then directly added to TRIzol reagent (Thermo Fisher #15596026); for cell lines media was removed and TRIzol reagent was added directly to the tissue culture dish. RNA was isolated from TRIzol reagent, treated with Turbo DNAse (Thermo Fisher #AM1907), and purified using RNA Clean and Concentrator-5 spin columns (Zymo #R1013) according to manufacturer's instructions.
Trizol reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Japan, Germany, United Kingdom, Canada, France, Italy, Australia, Spain, Switzerland, Netherlands, Belgium, Lithuania, Denmark, Singapore, New Zealand, India, Brazil, Argentina, Sweden, Norway, Austria, Poland, Finland, Israel, Hong Kong, Cameroon, Sao Tome and Principe, Macao, Taiwan, Province of China, Thailand
TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (5716 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4157 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (3871 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (3253 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2428 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2343 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2266 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2213 mentions)
Rneasy mini kit, by Qiagen (2160 mentions)
Sybr premix ex taq, by Takara Bio (2120 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1895 mentions)
Nanodrop, by Thermo Fisher Scientific (1858 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1718 mentions)
Primescript rt master mix, by Takara Bio (1544 mentions)
Dnase 1, by Thermo Fisher Scientific (1524 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1364 mentions)
Sybr premix ex taq 2, by Takara Bio (1351 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1337 mentions)
M mlv reverse transcriptase, by Promega (1155 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1153 mentions)
85 338 protocols using trizol reagent
1
RNA Isolation from Frozen Tissue
2
CXCL8 Expression in Mammary Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mammary epithelial cells from primary cultures were obtained as described elsewhere21 (link) and seeded in 24-well plates at 44,000 cells/well. After 24 h incubation at 37 °C and 5% CO2, wells were washed twice with warm PBS and 500 μl PBS or two doses (9 µg and 90 µg in 500 μl of PBS) of the solubilized and purified M-SAA3 were added to each well containing 500 μl of DMEM/F‐12 medium with 8 μg/ml bovine insulin and 50 μg/ml hydrocortisone by sextuplicate. After 3 h of incubation at 37 °C, 5% CO2, cells were gently washed with PBS and 500 μl of Trizol reagent (Thermo Fisher Scientific) were added to each well to collect and lysate the cells. The extraction of RNA was performed using the Trizol reagent (Thermo Fisher Scientific) and it was processed for qPCR analyses of CXCL8 expression as described previously21 (link). Relative gene expression was calculated using the 2∆Ct method with ACTB as reference gene.
3
RNA Extraction from Heart Tissues and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract RNA from heart samples (1 mL/30 mg of tissue), primary cardiomyocytes and HL-1 cell cultures, as previously described [15 (link),28 (link)]. Cell lysis was performed with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) (500 μL per well in a 24-well plate) after one washing step with PBS, while tissue samples were lysed with TissueLyser II (QIAGEN) in 1000 μL of TRIzol (Thermo Fisher Scientific, Waltham, MA, USA). Chloroform (200 μL per 1000 μL of TRIzol) was added to the homogenate to separate the RNA-containing aqueous phase. Thereafter, RNA was precipitated by adding an equal volume of isopropanol to the aqueous phase. Qualitative and quantitative RNA measurements were assessed both by spectrophotometer and Agilent 2100 Bioanalyzer. RNA showing an RNA integrity number higher than 7 was used for experiments.
4
mRNA Extraction: Diverse Differentiation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To collect mRNA, two different methods were used depending on the differentiation procedure. mRNA from cells that underwent osteogenic and adipogenic differentiation was isolated via a TRIzol reagent (Thermo Fisher Scientific), while the mRNA from cells that underwent chondrogenic differentiation was extracted by a Total RNA Kit I (Omega Bio-tek, VWR). Common elements to both protocols include the addition of 1 mL of the TRIzol reagent with glycogen (Thermo Fisher Scientific) to the cells, followed by the addition of 200 μL of chloroform (Sigma-Aldrich). The samples were vortexed, incubated, and then centrifuged per the manufacturer's instructions.
5
Biotin-Labeled circ_0004488 Probe Pulldown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RiboBio (China) created a biotin-labeled version of the circ_0004488 probe. Cervical cancer cells were fixed for 10 minutes in 1 percent formaldehyde (Sigma-Aldrich) before being lysed and sonicated. Following centrifugation, 20 μl of the supernatant was kept as input, while the remainder was incubated overnight with a biotin-labeled circ_0004488 probe-streptavidin M-280 bead combination (Thermo Fisher Scientific). To reverse the formaldehyde crosslinking, the beads-RNA combination was washed and treated with lysis solution and proteinase K (Sigma-Aldrich). Finally, the TRIzol reagent (Thermo Fisher Scientific) was added to the mixture for RNA extraction. The Magna RIP kit was used for the RNA immunoprecipitation experiment (Millipore, Billerica, MA, USA). RIP buffer containing IgG-antibody or Ago2-labeled magnetic beads was utilized, and the immunoprecipitated RNAs were then produced using the TRIzol reagent (Thermo Fisher Scientific).
6
ChIP-qPCR Analysis of Liver Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from isolated hepatocytes using the TRIzol reagent (15596018; Thermo Fisher Scientific) according to the manufacturer’s protocol. For snap frozen liver tissues, samples were homogenized in TRIzol reagent in a MP Biomedicals Lysing Matrix D tube (Thermo Fisher Scientific) using the Precellys 24 homogeniser (Bertin Technologies). 2μg of RNA was used to prepare complementary DNA (cDNA) using the Maxima First Strand cDNA Synthesis Kit (K1641; Thermo Fisher Scientific). ChIP was performed as described before [73 (link)] using 2μL of anti-NFκB p65 pS536 antibody (#3033; Cell Signaling Technology) per ChIP sample. DNA was then isolated from eluted samples using ChIP DNA Clean & Concentrator (D5205; Zymo Research) in 20μL volume. qPCR was then performed with the Maxima SYBR Green qPCR Master Mix (K0221; Thermo Fisher Scientific) using 10ng cDNA or 1uL of ChIP input/sample per reaction. Analysis was performed via the 2-ΔΔCt method [74 (link)] with Eef2 (for cDNA) or input (for ChIP) as the normalizing control. Negative control for ChIP was a gene desert in chromosome 15 [75 (link)]. Primer sequences are available in S4 Table .
7
Real-Time qRT-PCR for mRNA and miRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol reagent (Thermo Fisher SCIENTIFIC), and cDNA was synthesized from one µg of RNA using random primers and iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). mRNA levels were quantified using IQ SYBR green supermix (Bio-Rad Laboratories) on the CFX96 real-time system instrument (Bio-Rad). Specific primers located in different exons of the same gene were designed to detect relative mRNA levels. The housekeeping β-2 microglobulin or c-ABL genes were used for internal normalization. All the PCR reactions were performed in triplicate, and PCR products were also visualized on agarose gels after ethidium bromide staining. The oligonucleotide sequences are reported in Supplemental Table S2. Relative fold variations were calculated using the 2−ΔΔCt method by the formula: 2−(sampleΔCt − controlΔCt), where ΔCt is the difference between the amplification fluorescent thresholds of the gene of interest and the internal reference gene/s used for normalization [39 (link)].
To detect the levels of mature miR-128, total RNA was prepared with TRIzol reagent (Thermo Fisher SCIENTIFIC) and miR amounts were evaluated by using the TaqMan miRNA assay kit (Thermo Fisher SCIENTIFIC). For normalization of RNA levels, the amounts of small nucleolar RNA RNU6 (Thermo Fisher SCIENTIFIC) were measured [39 (link)].
To detect the levels of mature miR-128, total RNA was prepared with TRIzol reagent (Thermo Fisher SCIENTIFIC) and miR amounts were evaluated by using the TaqMan miRNA assay kit (Thermo Fisher SCIENTIFIC). For normalization of RNA levels, the amounts of small nucleolar RNA RNU6 (Thermo Fisher SCIENTIFIC) were measured [39 (link)].
8
Extracting Aortic Endothelial RNA from Mice
9
Transcriptional Response of G. pamelaeae to DOPAC Exposure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Method 1 (compound added at mid-exponential phase): Turbid 48 hr starter cultures of G. pamelaeae 3C grown in BHI medium were inoculated 1:100 into triplicate Hungate tubes containing 20 mL BHI medium with 10 mM formate. When cultures reached OD600 = 0.110, DOPAC (0.5 mM final) or vehicle (water) was added to the cultures. The cultures were then grown at 37°C anaerobically and harvested when they reached OD600 = 0.185. They were centrifuged, and cell pellets were re-suspended in 500 µL Trizol reagent (ThermoFisher, catalog#: 15596026).
Method 2 (compound added at the beginning of growth): Turbid 48 hr starter cultures of G. pamelaeae 3C grown in BHI medium were inoculated 1:100 into triplicate hungate tubes containing 20 mL BHI with 10 mM formate and DOPAC (0.5 mM final) or vehicle (water). These cultures were then left to grow at 37°C anaerobically. When cultures reached OD600 = 0.110, they were harvested. They were centrifuged, and cell pellets were re-suspended in 500 µL Trizol reagent (ThermoFisher, catalog#: 15596026).
RNA extraction and sequencing: this was performed using the exactly same setup as described above, except the reads were aligned to the genome of Gordonibacter pamelaeae 3C.
Method 2 (compound added at the beginning of growth): Turbid 48 hr starter cultures of G. pamelaeae 3C grown in BHI medium were inoculated 1:100 into triplicate hungate tubes containing 20 mL BHI with 10 mM formate and DOPAC (0.5 mM final) or vehicle (water). These cultures were then left to grow at 37°C anaerobically. When cultures reached OD600 = 0.110, they were harvested. They were centrifuged, and cell pellets were re-suspended in 500 µL Trizol reagent (ThermoFisher, catalog#: 15596026).
RNA extraction and sequencing: this was performed using the exactly same setup as described above, except the reads were aligned to the genome of Gordonibacter pamelaeae 3C.
10
Transcript Analysis of Nudt21 Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We dissected and hemisected the brains of five-week-old and dissected the hippocampi of 46-week-old Nudt21+/- mice and their wild-type littermates. We immediately flash froze the tissue in liquid nitrogen and stored it temporarily at −80°C. We later homogenized the tissue in TRIzol Reagent (ThermoFisher Scientific) with the Polytron PT 10–35 GT (Kinematica). We isolated RNA from the hemisected brains by chloroform phase separation, precipitated it with 2-propanol, and washed it with 75% ethanol. We eluted the purified RNA in water.
We extracted RNA from shRNA-infected, human ESC-derived neurons in a 12-well plate: four wells non-silencing scrambled (RHS4348) and eight wells shNUDT21 (V2LHS_253272 and V2LHS_197948) (Dharmacon). We lysed the neurons in the tissue-culture plate with TRIzol Reagent (ThermoFisher Scientific) and immediately transferred the lysate to microfuge tubes for trituration. We then isolated the RNA by chloroform phase separation, precipitation with 2-propanol, washing with 75% ethanol, and eluting in water as with the mice.
We extracted RNA from shRNA-infected, human ESC-derived neurons in a 12-well plate: four wells non-silencing scrambled (RHS4348) and eight wells shNUDT21 (V2LHS_253272 and V2LHS_197948) (Dharmacon). We lysed the neurons in the tissue-culture plate with TRIzol Reagent (ThermoFisher Scientific) and immediately transferred the lysate to microfuge tubes for trituration. We then isolated the RNA by chloroform phase separation, precipitation with 2-propanol, washing with 75% ethanol, and eluting in water as with the mice.
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!