RNA isolation, quantification, and quality control

HF Hernan P. Fainberg
MB Mark Birtwistle
RA Reham Alagal
AA Ahmad Alhaddad
MP Mark Pope
GD Graeme Davies
RW Rachel Woods
MC Marcos Castellanos
SM Sean T. May
CO Catharine A. Ortori
DB David A. Barrett
VP Viv Perry
FW Frank Wiens
BS Bernd Stahl
EB Eline van der Beek
HS Harold Sacks
HB Helen Budge
MS Michael E. Symonds
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For RNA isolation from each fat depot from 5 animals within each nutritional group, and a 100 mg was used from samples collected at 7, and 1000 mg from taken at 28 days of age, respectively. These were mixed with 2 ml of TRI reagent (Sigma-Aldrich). Total RNA was extracted using the RNeasy Plus kit (Qiagen) according to the manufacturer’s instructions and its quantity measured with a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Optical density ratios (260/280 nm) were >1.9 for all samples. Total RNA quality was assayed by the Agilent BioAnalyzer RNA 6000 Nano Kit (Agilent Technologies) and only used if distinct ribosomal peaks measured (i.e. RIN > 7).

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