RNA isolation and reverse transcription
Total RNA was prepared from LM using Trizol Reagent (TaKaRa,
Dalian, China) according to the instructions of the manufacturer. The
purity and concentration of total RNA were measured by a spectrophotometer
(Beckman Coulter, DU800) at 260 and 280 nm. Ratios of
absorption (260/280 nm) of all samples were between 1.8 and 2.0.
Aliquots of 4 μg RNA sampleswere subjected to electrophoresis through a 1.3% agarose-formaldehyde gel to detect their integrity.
For each sample, 2 μg of total RNA was used to synthesize the cDNA using a
PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China)
according to the instructions of the manufacturer.