RNA was isolated from samples collected in TRIzol (Invitrogen) using a chloroform-isopropanol protocol. 200μL chloroform was added to 1mL TRIzol samples, transferred to a heavy phase-lock gel tube (QuantaBio), and spun in a microcentrifuge at 12,000xg for 15 min at 4°C. The aqueous layer was transferred to a new tube containing 1μL of glycogen (Fisher Scientific) and equal volume isopropanol added. After 10 min of incubation at room temperature, sample was spun at 20,000xg for 20 min at 4°C, then RNA pellet was washed with fresh 75% ethanol before drying and resuspending in 40μL nuclease-free water. Isolated mRNA was DNase treated using Turbo DNase (Invitrogen) in reactions consisting of 11 μL (~15μg) RNA and 1.5μL Turbo DNase according to manufacturer’s protocol. DNase-treated RNA was purified using lithium chloride precipitation as described in mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen). 2μg RNA per sample was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen) and MPRA-specific primer (Table S7 , primer #2).
Trizol
Manufactured by Quantabio
TRIzol is a monophasic solution of phenol and guanidine isothiocyanate, designed for the isolation of total RNA from various biological samples. It is a widely used reagent for the extraction and purification of RNA.
Automatically generated - may contain errors
Lab products found in correlation
Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (2 mentions)
Glycogen, by Thermo Fisher Scientific (2 mentions)
Heavy phase lock gel tube, by Quantabio (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
2 protocols using trizol
1
RNA Isolation and cDNA Synthesis
2
RNA Isolation and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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RNA was isolated from samples collected in TRIzol (Invitrogen) using a chloroform-isopropanol protocol. 200μL chloroform was added to 1mL TRIzol samples, transferred to a heavy phase-lock gel tube (QuantaBio), and spun in a microcentrifuge at 12,000xg for 15 min at 4°C. The aqueous layer was transferred to a new tube containing 1μL of glycogen (Fisher Scientific) and equal volume isopropanol added. After 10 min of incubation at room temperature, sample was spun at 20,000xg for 20 min at 4°C, then RNA pellet was washed with fresh 75% ethanol before drying and resuspending in 40μL nuclease-free water. Isolated mRNA was DNase treated using Turbo DNase (Invitrogen) in reactions consisting of 11 μL (~15μg) RNA and 1.5μL Turbo DNase according to manufacturer’s protocol. DNase-treated RNA was purified using lithium chloride precipitation as described in mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Invitrogen). 2μg RNA per sample was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen) and MPRA-specific primer (Table S7 , primer #2).
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