Total RNA from the serum and tissue samples was isolated with RNAVzol LS (Vigorous Biotechnology Co, Beijing, China, http://www.vigorousbiol.com/ ) according to the manufacturer's protocol. The quality, quantity and integrity of the RNA were monitored using a NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
Rnavzol ls
Manufactured by Vigorous Biotechnology
Sourced in China
RNAVzol LS is a specialized laboratory reagent designed for the isolation and purification of ribonucleic acid (RNA) from various biological samples. Its core function is to facilitate the efficient extraction and preservation of high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Rnavzol, by Vigorous Biotechnology (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Diethyl pyrocarbonate treated water, by Solarbio (1 mentions)
One step primescript rt pcr kit, by Takara Bio (1 mentions)
Ab28478, by Abcam (1 mentions)
M mulv transcriptase, by New England Biolabs (1 mentions)
Sc 366, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Iq5 system, by Bio-Rad (1 mentions)
6 protocols using rnavzol ls
1
Total RNA Isolation from Serum and Tissue
2
RNA Extraction from Serum, Urine, and Tissue
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Total RNA was isolated from the serum samples (5 ml, collected in tubes containing EDTA) or urine or tissues using RNAVzol LS or RNAVzol (Vigorous Biotechnology Beijing Co., Ltd.) according to the manufacturer's protocol. The concentration and purity of the RNA samples were determined by measuring the optical density ratio at 260/280 nm.
3
RNA Isolation from Blood and Tissue
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Total RNA from whole blood samples or heart tissue samples was isolated using RNAVzol LS or RNAVzol (both Vigorous Biotechnology Beijing Co., Ltd.), respectively, according to the manufacturer's protocol to isolate small RNAs. Briefly, 500 µl whole blood samples or 10 mg heart tissue samples were mixed with 500 µl RNAVzol LS or RNAVzol, respectively. The mixture was incubated at room temperature for 15 min. Then, 200 µl chloroform was added and incubated with the above mixture for 5 min. After that, the mixture was centrifuged at 11,000 × g for 10 min at 4°C. The supernatant was collected and 500 µl dimethyl carbinol was added. The supernatant mixture was then incubated at 4°C overnight. After that, the mixture was centrifuged at 11,000 × g for 10 min at 4°C and the precipitate was retained. Finally, 10 µl diethyl pyrocarbonate-treated water (Beijing Solarbio Science and Technology Co., Ltd.) was added to dissolve the precipitate. The quality, quantity and integrity of RNA were monitored using a NanoDrop spectrophotometer (ND-1000; Thermo Fisher Scientific, Inc.).
4
RT-PCR Analysis of Ferroptosis in SKOV3 and Hey Cells
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SKOV3 and Hey cells were seeded in 6-well plates at a density of 10,000 cells/well overnight at 37°C. Then, SKOV3 and Hey cells were treated with 100 and 200 µg/ml CMP for 24 h at 37°C in the presence or absence of ferrostatin-1 (Fer-1, MCE). Total RNA was isolated from SKOV3 and Hey cells using RNAVzol LS (Vigorous Biotechnology Beijing Co., Ltd.) according to the manufacturer's protocol. The concentration and purity of RNA samples were determined by measuring the optical density (OD) 260/OD280.
RT-PCR was performed using a Takara PrimeScript One Step RT-PCR kit (Takara Bio, Inc.) according to the manufacture's protocols with three experiments replicated. The PCR amplifications were performed in a 50-µl reaction system containing 20 µl RNase Free ddH2O, 25 µl 2X Step Buffer, 2 µl PrimeScript 1 Step Enzyme Mix, 1 µl upstream primer (1 µM), and 1 µl downstream primer (1 µM). The PCR was as follows: 50°C for 30 min; 94°C for 2 min; and 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min. GAPDH was used as an internal control using the 2−∆∆Cq method (17 (link)). The primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome ) and the sequences were listed in Table I .
RT-PCR was performed using a Takara PrimeScript One Step RT-PCR kit (Takara Bio, Inc.) according to the manufacture's protocols with three experiments replicated. The PCR amplifications were performed in a 50-µl reaction system containing 20 µl RNase Free ddH2O, 25 µl 2X Step Buffer, 2 µl PrimeScript 1 Step Enzyme Mix, 1 µl upstream primer (1 µM), and 1 µl downstream primer (1 µM). The PCR was as follows: 50°C for 30 min; 94°C for 2 min; and 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min. GAPDH was used as an internal control using the 2−∆∆Cq method (17 (link)). The primers were designed using Primer-BLAST (
5
Whole Blood RNA Extraction
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The total RNA from the whole blood samples (5 ml) collected in tubes containing EDTA was extracted with RNAVzol LS (Vigorous Biotechnology, Beijing) rigorously according to the manufacturer's instructions. The concentration and the purity of the RNA samples were assayed by absorbent density analysis on OD 260 /OD 280 .
6
Quantifying miRNAs and Protein Expression
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The total RNA from the whole blood samples (5 ml) collected in tubes containing EDTA was rigorously extracted with RNAVzol LS (Vigorous Biotechnology, Beijing) according to the manufacturer's instructions. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) was conducted with the samples to detect and quantify mature miRNAs by using stem-loop antisense primer mix and M-MuLV transcriptase (NEB).
The SYBR Green I method was used for real-time PCR with the Bio-Rad iQ5 system (Bio-Rad) according to the manufacturer's instructions (TaKaRa) The relative expression level of a miRNA was normalized to an internal invariant control, U6 small nucleolar RNA. Each reaction was performed in triplicate, and analysis was performed using the 2 -△△CT method.
Western blot analysis A total of 15 µg protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane (Millipore), followed by 8% nonfat dry milk blocking. After washing with PBST three times (5 min/wash), the membranes were incubated with primary antibodies at 4°C overnight. The blot was incubated with HRP-conjugated anti-IgG, followed by detection with ECL (Millipore). Antibodies against LXRα (ab28478) were purchased from Abcam. FAS (#3180) and β-actin (#4970) were obtained from Cell Signaling Technology. An antibody against SREBP1 was purchased from Santa Cruz Biotechnology (sc-366).
The SYBR Green I method was used for real-time PCR with the Bio-Rad iQ5 system (Bio-Rad) according to the manufacturer's instructions (TaKaRa) The relative expression level of a miRNA was normalized to an internal invariant control, U6 small nucleolar RNA. Each reaction was performed in triplicate, and analysis was performed using the 2 -△△CT method.
Western blot analysis A total of 15 µg protein was separated by 10% SDS-PAGE and then transferred to PVDF membrane (Millipore), followed by 8% nonfat dry milk blocking. After washing with PBST three times (5 min/wash), the membranes were incubated with primary antibodies at 4°C overnight. The blot was incubated with HRP-conjugated anti-IgG, followed by detection with ECL (Millipore). Antibodies against LXRα (ab28478) were purchased from Abcam. FAS (#3180) and β-actin (#4970) were obtained from Cell Signaling Technology. An antibody against SREBP1 was purchased from Santa Cruz Biotechnology (sc-366).
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