Resource owners then learn the value of the shock, and the owners choose extraction quantities.
When the decay of the gas-extraction quantity occurs, the gas extraction enters the transitional period.
Following RNA extraction, the quantity and quality of extracts may be measured [ 26, 39, 52].
After DNA extraction, the quantity and purity of the DNA was assessed by spectrophotometry at 260/280 nm and by 1% agarose electrophoresis.
After extraction, RNA quantity was measured using a Nanodrop spectrophotometer (Nanodrop Technologies, Oxfordshire, UK). 8 μg of total RNA from each batch were first reverse transcribed to single-stranded cDNA using an oligo d(T 24 primer (Sigma) and Superscript III reverse transcriptase (Invitrogen).
Following extraction, RNA quantity was determined fluorometrically using a Qubit® 2.0 fluorometer with the broad-range total RNA assay kit (Life Technologies and RNAA integrity was visually checked with ultraviolet light after gel electrophoresis of 1 µg of RNA in 1.5 % agarose gels stained with SYBR® Green (SYBR® safe gel stain; Life Technologies).
For each extraction, a quantity of ground tooth (ca. 0.2 g) was incubated for 24h at 50°C with shaking (220 rpm/min), in 4 ml of an incubation buffer consisting of 0.5M EDTA, 0.5% SDS and 3 mg/ml proteinase K. Afterwards, the DNA was purified using the QIAquick PCR Purification Kit (Qiagen, Germany), according to the manufacturer's protocol.
After extraction, quality and quantity of yielded DNA were evaluated using a spectrophotometer (NanoDrop ND-1000, Thermo Scientific) and then stored at −20°C until PCR reaction.
The majority of the time for Option 2 (67% of the extractions), the quantity averaged 77 μg of RNA from only 100 mg of tissue in the first extraction attempt, so no pooling was needed.
