TRIzol RNA extraction method.

(i)
Reagents
1.
1. TRIzol reagents.
2. Chloroform
3. Isopropyl alcohol
4. 75% ethanol (DEPC H2O preparation)
5. DEPC H2O
(II) Operation step
1. Sample treatment:
(1)
culture
cells: harvest cells 1-5×107, move into 1.5 ml
centrifuge tube
, add 1 ml Trizol, mix well, set at room temperature 5min.
(2)
tissue
: take 50-100 mg of tissue (fresh or -70 degrees C and liquid nitrogen preserved tissue can be) placed in 1.5 ml centrifugal tube, add 1 ml Trizol full homogeneity, room temperature sit 5min.
2. Add 0.2 ml chloroform, oscillate 15s and set aside 2min.
3. Centrifuges at 4 degrees C, 12000g× 15min, cleared.
4. Add 0.5 ml of isopropyl alcohol, gently mix the liquid in the tube and set aside at room temperature for 10min.
5. Centrifuges at 4 degrees C, 12000g× 10min, discarded.
6. Add 1 ml of 75% ethanol and gently wash the precipitation. 4 degrees C, 7500g×5min, discarded.
7. Dry and add the appropriate amount of DECC H2O dissolved (65 degrees C solubility 10-15min).
(iii) Precautions
1. Sample volume and Trizol addition must be in accordance with step (1) ratio, can not arbitrarily increase the sample size or reduce trizol volume, otherwise the endo endo-rooted RNase inhibition is incomplete, resulting in RNA degradation.
2. The experimental process must strictly prevent contamination of RNsae.
total RNA quantitative
. The RNA quantification method is similar
the
of dna. RNA has the largest absorption peak at a wavelength of 260nm. As a result, RNA concentrations can be measured with 260nm wavelength dlight, with an OD value of 1 equivalent to about 40 μg/ml of single-stranded RNA. If you use a 1cm optical diameter, dilute the DNA sample with ddH2O n times and ddH2O as a blank control, according to the value of OD260 read at this time can calculate the concentration before the sample dilution:
RNA (mg/ml) - 40×OD26 The ratio of OD260/OD280 for 0 readings × dilution multiply (n)/1000
RNA pures is 2.0, so the purity of RNA can be estimated based on the ratio of OD260/OD280. A lower ratio indicates the presence
residual
protein, and a high ratio indicates degradation of RNA.
.