Total mRNA was extracted using the RNAeasy Minikit (Qiagen, Crawley, UK) according to the manufacturer’s protocol. Briefly, EC were collected from the transwell co-culture filters using accutase, lysed and then added to a column. After three washes, mRNA was eluted from the column with water. mRNA concentration was measured using NanoDrop spectrofluorimeter (LabTech) and mRNA was stored at −80 °C. To convert mRNA to cDNA, random primers (Promega, USA) were annealed to 1 μg of mRNA for 5 minutes at 70 °C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1× Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37 °C for 1 h, followed by 5 minutes at 95 °C. To analyse mRNA, FAM-labeled E-selectin and SOCS3 primers and VIC-labelled 18S primers, were bought as Assay on Demand kits from Applied Biosystems (Warrington, UK). Samples were amplified in duplicates using the 7500HT real-time PCR machine (Applied Biosystems) and analysed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2–ΔΔCt method).
Superscript buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Superscript Buffer is a specialized solution designed for use with Superscript enzyme products. It provides optimal buffer conditions for reverse transcription reactions. The buffer composition ensures efficient and reliable performance of the Superscript enzyme during the cDNA synthesis process.
Automatically generated - may contain errors
Lab products found in correlation
Rnaeasy mini kit, by Qiagen (4 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Random primer, by Promega (4 mentions)
Sds 2, by Thermo Fisher Scientific (4 mentions)
7500 ht real time pcr machine, by Thermo Fisher Scientific (4 mentions)
Dntps, by Promega (4 mentions)
Nanodrop spectrofluorimeter, by EQT (3 mentions)
Out rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Ivt labeling kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
6 protocols using superscript buffer
1
Endothelial Cell mRNA Extraction and Analysis
2
Quantifying mRNA Expression Levels
3
Quantifying mRNA Expression Levels
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Total mRNA was extracted using the RNAeasy Minikit (Qiagen, Crawley, UK) according to the manufacturer’s protocol. Briefly, PBMC were first lysed, then added to a column, after three washes, mRNA was eluted from the column with water. mRNA concentration was measured using Nanodrop spectrofluorimeter (LabTech) and mRNA was stored at −80°C. To convert mRNA to cDNA, random primers (Promega, Maddison, USA) were annealed to 1 μg of mRNA for 5 minutes at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U Superscript II Reverse Transcriptase (RT), 10 U RNAout RNase inhibitor, 1X Superscript Buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37°C for 1 hour, followed by 5 minutes at 95°C. To analyze mRNA, FAM-labelled SPHK1, SPHK2 and SPNS2 primers and VIC-labelled 18S primers were bought as Assay on Demand kits from Applied Biosystems (Warrington, U.K.). Samples were amplified in duplicates using the 7500HT Real-Time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2^−deltadelta Ct method).
4
Microarray Analysis of Cultured Cells
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Total RNA was extracted from cultured cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA quantity and quality was assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Only samples with an RNA integrity number average of 7–10 were used. For microarray analyses, the RNA was purified and amplified twice using the SenseAmp RNA Amplification kit (Ambion, Austin, TX, USA). The sense RNA was then labelled with the biotin-labeled antibody from the IVT Labeling kit (Affymetrix Inc., Santa Clara, CA, USA), followed by double stranded-cDNA clean up using Sample Cleanup Module (GeneChip; Affymetrix, Inc.) and fragmented RNA using fragmentation buffer (Qiagen, Inc., Valencia, CA, USA). The remaining RNA was reverse transcribed (RT) to cDNA for validation by quantitative polymerase chain reaction (qPCR). cDNA was generated in a 25 µl reaction volume using 2 ng of total RNA, Superscript III (0.2 µl/reaction), random hexamers (9 µg/reaction), 5 mM deoxynucleotides (0.5 µl/reaction) and 1X Superscript buffer (Invitrogen; Thermo Fisher Scientific, Inc.).
5
RNA Isolation and Reverse Transcription
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RNA was isolated using Trizol reagent (Invitrogen 15596-026) following the manufacturer’s instructions. Samples were either stored at −80 °C (for a long-term storage) or used for reverse transcription (RT). For RT, 1 μl of dNTPs (ThermoScientific R0141, R0151, R0161, R0171) was mixed with 1 μl of 20 μM oligo dT, 3 μg of RNA and H2O to a final volume of 12 μl (reaction mix 1). The reaction mix 1 was heated at 65 °C for 5 min (denaturation step) and chilled on ice. In parallel, 4 μl of Superscript buffer (SuperScript II Reverse Transcriptase Invitrogen, cat. # 18064-022) was mixed with 2 μl of 0.1M DTT, 1 μl of Ribonuclease inhibitor and 0.5 μl of Superscript Reverse Transcriptase (SuperScript II Reverse Transcriptase Invitrogen, cat. # 18064-022) (reaction mix 2). The reaction mix 1 was well mixed with the reaction mix 2 and incubated at 44 °C for 1 h (reverse transcription) followed by heat inactivation (70 °C for 15 min).
6
mRNA Extraction and Quantification Protocol
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Total mRNA was extracted using the RNAeasy mini kit (Qiagen, Crawley, U.K.) according to the manufacturer’s protocol. Briefly, EC were collected from the Transwell coculture filters using Accutase, lysed, and then added to a column. After three washes, mRNA was eluted from the column with water. mRNA concentration was measured using a NanoDrop spectrofluorometer (LabTech) and mRNA was stored at −80°C. To convert mRNA to cDNA, random primers (Promega) were annealed to 1 μg of mRNA for 5 min at 70°C, after which the following mastermix was added to give a final volume of 30 μl: 10 U of SuperScript II reverse transcriptase, 10 U of RNaseOUT, 1× SuperScript buffer (all from Invitrogen) and 10 mM dNTPs (Promega). The reaction was run at 37°C for 1 h, followed by 5 min at 95°C. To analyze mRNA, FAM-labeled E-selectin and suppressor of cytokine signaling (SOCS)3 primers and VIC-labeled 18S primers were bought as Assays-on-Demand kits from Applied Biosystems (Warrington, U.K.). Samples were amplified in duplicates using the 7500HT real-time PCR machine (Applied Biosystems) and analyzed using the software package SDS 2.2 (Applied Biosystems). Data were expressed as relative expression units relative to 18S or as fold change (2−ΔΔCt method).
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