For FDD gene expression analysis, as well as the use of any other RNA-based gene expression technique, contaminating genomic DNA must be removed prior to reverse transcription synthesis of the single-stranded cDNA and subsequent PCR operations.This experiment was derived from PCR Laboratory Guidelines (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Genomic DNA removal experiment from total RNA
Materials and Instruments
Denaturing (formaldehyde) agarose gel Agarose Distilled water Formaldehyde Distilled water Ethanol Formaldehyde MOPS buffer Ethylenediaminetetraacetic acid MOPS Sodium acetate Phenol Chloroform (3:1) solution Chloroform Liquid Phenol Tris-HCl Electrophoresis Buffer Move I. Materials For more product details, please visit Aladdin Scientific website.
Centrifuges and rotors Centrifuge tubes Dispensing plates Microwave oven
1. Buffers, solutions and reagents
Denaturing (formaldehyde) agarose gel (see step 18 for preparation)
1 g agarose
83 ml distilled water
12.3 mol/L formaldehyde
Distilled water
Ethanol, 100%
Ethanol (70%), prepared with DEPC-treated H20
12.3mol/L (37%) formaldehyde, pH>4.0
10XMOPS buffer
0.01mol/L ethylenediaminetetraacetic acid (EDTA)
0.2mol/LMOPS
0.05mol/L sodium acetate
Phenol/chloroform (3:1) solution
10 ml chloroform
30 ml liquid phenol
10 ml Tris-HCl, pH 7.0
Electrophoresis buffer (see step 18f.)
10 ml10XMOPS
Dilute to 1X concentration with 900 ml distilled water
2. Nucleic acids and oligonucleotides
RNA from Scheme 1
3. centrifuges and rotors
Microfuge (recommend MocroSpin24, SorvallInstruments, Wilmington, DE)
4. Specialized equipment
1.5 ml centrifuge tube
Plate
microwave oven
5. Additional reagents
MessageClean DNA Removal Kit (GenHunter M601), including 10X reaction buffer, RNAase-free DNA enzyme I (10 U/ul), 3 mol/L sodium acetate, diethyl pyrocarbonate (DEPC)-treated H20, RNA, and RNA.
Spotting solution
II.
1. Digestion of total RNA with DNAase I. 2.
(1) If necessary, dilute the desired amount of RNA to be digested (up to 50ug) with DEPC-treated H20 in a maximum volume of 50ul.
(2) In a 1.5 ml centrifuge tube, add the following components in sequence (total reaction volume is 56.7ul).
Total RNA50ul
10X reaction buffer 5.7ul
DNA Enzyme I (10U/ul) 1.0ul
(3) Mix gently and incubate at 37°C for 30 min.
2. Extraction and ethanol precipitation of DNA-free RNA
(4) Heat phenol crystals to 65°C in the glass storage container provided at the time of purchase to melt and use to prepare the phenol/chloroform solution.
(5) Add 30 ml of melted phenol to 10 ml of chloroform and mix well.
(6) Add 10 mlpH7.0 of Tris-HCl and mix well to form a saturated phase before use.
(7) To each 1.5 ml centrifuge tube for the DNAase I reaction, add 40 ul of phenol/chloroform solution and vortex for 30 s.
(8) Place the solution on ice for 10 min.
(9) Centrifuge at 4°C for 5 min at maximum speed.
(10) Collect the supernatant and transfer it to a clean, labeled 1.5 ml centrifuge tube.
(11) Add 5ul of 3mol/L sodium acetate and 200ul of 100% ethanol and mix well.
(12) Allow the mixture to stand at -80°C for more than 1 hour.
(13) Centrifuge at 4°C for 10 min at maximum speed to precipitate RNA.
(14) Carefully remove the supernatant and wash the RNA precipitate with 0.5 ml of 70% ethanol (prepared with DEPC-treated solution). Do not stir up the precipitate.
(15) Centrifuge at 4°C for 5 min at maximum speed and remove the supernatant. Centrifuge again briefly to remove any residual liquid, but do not stir the precipitate.
(16) Resuspend the RNA with 10-20ul of DEPC-treated H2O.
3. RNA fixation and integrity testing
(17) Dilute the DNA-free RNA sample with distilled water at 1:1000 and measure OD260.
(18) Prepare a denaturing (formaldehyde) agarose gel according to the following protocol.
a. Add the following ingredients to a container that can be used in a microwave oven.
10XMOPS 10 ml
Agarose 1 g
Distilled water 83 ml
b. Microwave for about 3 min to melt the agarose.
c. Cool agarose to below 50°C.
d. Add 7 mll 2.3 mol/L (37%) formaldehyde solution and mix gently.
e. Pour the gel into the prepared dispensing plate and reposition the comb.
The gel is then mixed into the prepared plate. f. Prepare electrophoresis buffer (1L) by diluting 100 ml of 10XMOPS to a concentration of 1X with 900 ml of distilled water. Submerge the agarose gel with the electrophoresis buffer.
(19) Dissolve 2 to 3ug of DNAzyme pre-digested and post-digested RNA samples according to the following protocol, mix with the RNA spotting mix, and test for RNA integrity on a 7% formaldehyde agarose gel.
a. Take 1~10ul (2~3ug) of RNA, add to 20ul of RNA Spotting Mix and mix in a 1.5 ml centrifuge tube. b. Add to the RNA Spotting Mix.
b. Incubate at 65°C for 10min.
c. Centrifuge briefly to collect condensate.
d. Place sample on ice for 5 min.
e. Add all of the sample to the RNA gel.
f. Run at 50-60V for approximately 45 min, or until ribosomal subunits are resolved.
(20) On denaturing gels, ribosomal subunits will appear as distinct bands. Degraded RNA will appear as a diffuse patch during extraction or DNAase treatment. The size of the ribosomal subunits varies depending on the organism being analyzed.