RAW cells were harvested in a defined order by aspirating media from the 6-well dish and directly lysing cells by addition of 1 mL Trizol (Life technologies, Calsbad, CA). Lysed cells were immediately frozen at -80 °C. Samples were extracted in the same order as harvest order. Cell lysates were thawed and half the sample was subjected to RNA extraction. Samples were brought up to 1000 μL by addition of Trizol and then combined with 200 μL 1-bromo-3-chloropropane (Sigma-Alrich, St. Louis, MO). Samples were mixed using Genie vortex mixer for 15 s and centrifuged at 16,000 × g for 15 min at 4 °C. Aqueous phase was removed and passed through a Qiagen QiaShredder column to fragment any remaining gDNA in the sample. To each sample, an equal volume of Qiagen RNeasy RLT buffer with 1% β-mercaptoethanol and 70% ethanol was added and RNAs were extracted using the Qiagen AllPrep DNA/RNA mini kit system as described by manufacturer (Qiagen, Valencia, CA) including on-column Dnase I treatment. All sample processing was performed with amplicon free pipets and reagents in an amplicon free area to avoid contamination. RNA purity was determined by spectrophotometry. RNA integrity was analyzed using Agilent 2100 Bioanalyzer and calculated as an RNA integrity number (RIN) (Agilent Technologies, Santa Clara, CA).
Rneasy rlt buffer
Manufactured by Qiagen
Sourced in United States, Germany
The RNeasy RLT buffer is a lysis buffer used in the RNeasy kit from Qiagen for the isolation and purification of total RNA from a variety of sample types. The buffer aids in the disruption and lysis of cells or tissues to release the RNA, and it also contains guanidine thiocyanate to help inactivate RNases and ensure the integrity of the extracted RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Qiashredder column, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
1 bromo 3 chloropropane, by Merck Group (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
One step cell separation, by BD (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Quantasoft analysis pro software, by Bio-Rad (1 mentions)
Prism 9, by GraphPad (1 mentions)
8 protocols using rneasy rlt buffer
1
RNA Extraction from RAW Cells
2
RNA Extraction from Gels
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Gels were transferred to new six-well non-TC plates, taking care to cause minimal mechanical disruption. RNeasy RLT buffer (QIAGEN) with 1:100 β-mercaptoethanol was added to the center of the gel at 350 μl per gel. Several minutes later, RLT buffer on each gel was gently pipetted up and down and collected. RNA was isolated with the RNeasy Micro Kit (QIAGEN) according to the manufacturer’s instructions.
3
Isolation and Lysis of PBMCs
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Blood was collected from healthy controls and patients in CPTTM tubes designed for one-step cell separation (Becton Dickinson, Mountain View, CA, USA). The sample was then immediately mixed and centrifuged at 1,800 RCF at ambient temperature for 30 min. The PBMC cell layer was then transferred to a 15 ml tube, and PBMCs were washed twice with PBS and lysed in RNeasy RLT buffer (Qiagen, Valencia, CA, USA).
4
RNA Extraction from Gels
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Gels were transferred to new six-well non-TC plates, taking care to cause minimal mechanical disruption. RNeasy RLT buffer (QIAGEN) with 1:100 β-mercaptoethanol was added to the center of the gel at 350 μl per gel. Several minutes later, RLT buffer on each gel was gently pipetted up and down and collected. RNA was isolated with the RNeasy Micro Kit (QIAGEN) according to the manufacturer’s instructions.
5
VEGF Expression Profiling in PAH Patients
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Forty samples of peripheral blood mononuclear cells (PBMCs) were purified from PAH patients and healthy controls, respectively. In short, blood was collected from patients and healthy subjects using one-step cell separation (Becton Dickinson, Mountain View, CA, USA). The samples were vortexed and then centrifuged at 1800 reactive centrifugal force at ambient temperature for 30 minutes. The PBMC cell layer was transferred to a 15-mL tube. After that, the PBMCs were washed twice with phosphate buffer saline and lysed in RNeasy RLT buffer (Qiagen, Valencia, CA, USA). Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and then reversely transcribed into complementary DNA using oligo primer and Superscript II (Invitrogen). The relative mRNA levels of VEGF were detected on the ABI Prism 7500 sequence detection system (Applied Biosystems) based on the SYBR-Green method. The β-actin was used as an internal reference gene. The primers used for VEGF were 5'-CCTCGCAGTCCGAGCCGGA-3' (forward) and 5'-GACCCAAAGTGCTCCTCGAAG-3' (reverse) and for β-actin were 5'-GGCGGCACCACCATGTACCCT-3' and 5'-AGGGGC CGGACTCGTCATACT-3'. Relative quantification of VEGF mRNA was performed according to the 2-ΔCt method.16 (link) All assays were executed in triplicate, at least three times independently.
6
Tumor Enrichment and RNA Extraction
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Frozen tumor blocks were manually dissected to remove muscle and stroma and to enrich for cellular areas containing ≥ 70% cancer cells and then cut into 10 μm sections. Normal colon was dissected from an area at least 8 cm away from the tumor, manually dissected to enrich for mucosa, and then cut into 10 μm sections. All slides were reviewed by a pathologist to outline areas of interest. Frozen sections were placed into RNeasy RLT buffer (Qiagen, Valencia, CA) and stored at − 80 °C. After completing the RNA extraction, RNA was quantitated by nanodrop (Thermo Scientific, Wilmington, DE).
7
Quantifying SARS-CoV-2 RNA in Cells
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Total RNA from HEK293T SARS-CoV-2 reporter cells treated as above was extracted following lysis in RNeasy RLT buffer (Qiagen) supplemented with 2-mercaptoethanol (10 μl/ml) and purified using RNeasy kits (Qiagen, Germany with on-column DNase digestion according to the manufacturer’s instructions, and additional RPE washes in order to fully remove FANA oligos (see ref. 31 for a discussion of the necessity of this precaution), and reverse transcribed to generate cDNA templates for ddPCR reactions as in 'Determination of XNAzyme activity on ex vivo SARS-CoV-2 genomic RNA'. Quantification of SARS-CoV-2 RNA (ORF7b and N2 sites) was performed in triplicate and normalised to a host reference gene, eIF2B2, using a singleplex assay: 0.9 μM primers ('eIF2B2_Fw' and 'eIF2B2_Rev') and 0.5 μM probe ('eIF2B2_Probe') were used with the following cycling conditions: 95 °C 10 min, 40 x [94 °C for 30 s, 58 °C for 1 min (with 2 °C/s ramp rate)], 98 °C for 10 min. Data were analysed using Quantasoft Analysis Pro software (Bio-Rad Laboratories, USA) and Prism 9.2.0 (Graphpad Software, USA). Statistical significance was determined using a paired t-test.
8
Co-cultivation of IVD Cells with C. acnes
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5 × 105 IVD cells in 3 mL media (approximately 1.7 × 105 cells/mL) were seeded onto 6-well plates and incubated for 24 h in DMEM/F12 supplied with 10% FCS but without A/A to allow co-cultivation with C. acnes. The bacteria were grown in liquid culture for 48 h before they were added to the IVD cells in a 100:1 multiplicity of infection (MOI).
To guarantee an immediate contact between the cells and the bacteria, the plates were centrifuged at 250× g for 5 min. The cultures were harvested 24 h post infection. The supernatants were sterile filtered and frozen at −80 °C for further ELISA experiments, whereas the pellets were washed three with Phosphate Buffered Saline (PBS, pH 7.4, 10010-015, Gibco, Carlsbad, CA, USA) to get rid of all the bacteria. The IVD cells were lysed with Qiagen RNeasy RLT buffer (74106, Qiagen, Hilden, Germany).
To guarantee an immediate contact between the cells and the bacteria, the plates were centrifuged at 250× g for 5 min. The cultures were harvested 24 h post infection. The supernatants were sterile filtered and frozen at −80 °C for further ELISA experiments, whereas the pellets were washed three with Phosphate Buffered Saline (PBS, pH 7.4, 10010-015, Gibco, Carlsbad, CA, USA) to get rid of all the bacteria. The IVD cells were lysed with Qiagen RNeasy RLT buffer (74106, Qiagen, Hilden, Germany).
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