RNA extraction from formalin-fixed, paraffin-embedded tissues
Formalin-fixed, paraffin-embedded tissues from the pathology department can be used for RNA expression analysis. RNA isolated from biopsy, surgical, and cadaveric tissues is not completely degraded after conventional fixation and paraffin embedding. Moreover, even if the tissue is not fixed immediately after excision, RNase-rich organelles such as the pancreas still contain RNA fragments of about 100 bases or more. A method for extracting RNA from 6 to 8 um tissue sections is described below.
Operation method
RNA extraction from formalin-fixed, paraffin-embedded tissues
Materials and Instruments
R N A Digestive solution β-mercaptoethanol Protease K (20 mg ml) Water balance Phenol Chloroform Glycogen TE Buffer Isopropyl alcohol Anhydrous ethanol Ethanol DEPC Water Move I. Materials and equipment Caveat 1) Guanidine thiocyanate is not toxic. Plants should be handled in a fume hood and stored in a brown bottle at room temperature for 2 months.2) High concentrations of proteinase K are required.3) Proteinase K and protein hydrolysis residues must be removed to avoid interference with subsequent steps. When extracting RNA with phenol: chloroform. Proteins are located at the interface between the organic and aqueous layers, while RNA is located in the aqueous phase.4) RNA from paraffin-embedded tissues extracted by this method is highly degraded, with fragment lengths ranging from 100 to 200 bases, and the degree of degradation varies from specimen to specimen depending on the fixation and paraffin-embedding conditions. For more product details, please visit Aladdin Scientific website.
(1) RNA digestion solution: each 100ml contains 40ml 4mol/L thiocyanic guanidine, 3mllmol/L Tris-HC (PH7.6),2.4ml 30% N-dodecylsarcosine sodium, DEPC treated water (54.6ml).
(2) β-mercaptoethanol: it must be added to the digestive solution before use, every l00ul solution plus 0.28ul 4 ℃ storage
(3) Proteinase K (20mg/ml): dissolved in DEPC water (50%) and glycerol (50%), stored at 20℃.
(4) water balance phenol: very sensitive to light and oxidation, in order to reduce the rate of oxidation of phenol, add 8-hydroxyquinoline (0.1%) phenol / water in the storage solution can be used in dark bottles stored at 4 ℃, the storage solution must be placed in the storage at 20 ° C, the expiration date of 2 years
5) Chloroform
(6) Glycogen: 1.0 mg/ml dissolved in water, stored in portions at 20°C.
7) TE buffer: 10 mmol/L Tris-HCl (PH8.0), 1 mmol/L EDTA (PH8.0)
8）Isopropyl alcohol
(9) Ethanol: anhydrous ethanol, 95% ethanol, 75% ethanol
10) DEPC water
Operation Methods
1. Paraffin section
Sections of 6-8 um thickness are made with a standard slicer. It is not necessary to change the knife for each specimen because the paraffin itself cleans the blade of the knife during each section. After cutting each specimen, use xylene to remove the paraffin residue from the blade and place the sections in 1.5 ml tubes. For easier handling of the sections, it is best to use rolled-up sections, which can be obtained by placing the paraffin blocks in a refrigerator to lower the temperature before sectioning.
2. Dewaxing
The first step in extracting the tissue is to dewax it with an organic solvent such as xylene, which must then be washed with ethanol to completely remove the xylene, which interferes with the protease in subsequent stages.
1) For each 1.5 ml Eppendorf tube, put up to 5 slices, the thickness of the slices is 6~8um, and use lml xylene to dewax at room temperature for 5 min, twice. Centrifuge the slices at high speed for lOmin, and keep the precipitate each time. At this time, it should be noted that the precipitate does not adhere to the bottom of the tube very tightly, and the supernatant should be removed carefully so as not to lose the tissue fragments.
2) Wash with 1 ml of anhydrous ethanol for 10 min, wash again with 95% ethanol, centrifuge for lOmin, and keep the precipitate.
3) Leave the tube open at 37℃ for 30 min to remove ethanol and dry the tissue in air.
3. Protein digestion
In order to obtain purified RNA, the tissue proteins must be removed, and this is best accomplished by digestion with protein hydrolases.
1) Add 1x the volume (100-300ul depending on the amount of tissue section) of digestion solution containing β-mercaptoethanol (0.28ul/100ul solution) to each kilodried sample.
2) Add Proteinase K to a final concentration of 6 mg/ml.
3) Incubate at 45℃ overnight.
4. RNA extraction
1) Add 1 times the volume of water-saturated phenol:chloroform to each tube, ratio 7:3.
2) Mix well, place on ice for 15 min12000 g centrifugation for 20 min.
3) Keep the upper aqueous phase and transfer it to another tube.
5. RNA Precipitation
RNA precipitation is obtained from the solution by precipitation with isopropanol using glycogen as a carrier.
1) Add 5ul of glycogen (1 mg/ml) and 1x Hoven's isopropanol to the aqueous phase and place at -20℃ for 48 h. To obtain a sufficient amount of RNA precipitation, it is necessary to leave it at 20℃ for a longer period of time.
2) Centrifuge at 12000 g at 4°C for 20 mm. The RNA precipitate does not adhere to the bottom of the microcentrifuge tube as well as the DNA precipitate, so it should be poured out gently. Be careful not to lose the RNA precipitate.
3) Wash the precipitate with 100ul of 75% ethanol and store at -20℃.