Expression experiments of multidrug resistance genes
Expression experiments of multidrug resistance genes
Expression experiments of multidrug resistance genes are mainly used for tumor therapy research.
Operation method
PCR amplification
Principle
Overexpression of the MDR-1 gene and its gene product, P-glycoprotein, occurs on the membrane of most MDR cells. Multidrug resistance gene expression assays were performed to detect overexpression of these specific genes by reverse transcription and PCR.
Materials and Instruments
RNA Move I. Total cellular RNA extraction Caveat 1. Operate at low temperatures whenever possible;2. use Trizon, etc. at appropriate discretion;3. Remove proteins and insoluble matter after each centrifugation.Wear gloves and a mask to prevent the effects of DNA enzymes during extraction. 4;4. Be aware that Trizon is corrosive;5. The most important thing is to prevent the contamination of DNase, gloves, masks are indispensable, and the guns and EP tubes used should be special for RNA extraction and free of DNA enzyme. For more product details, please visit Aladdin Scientific website.
PBS Chloroform Isopropyl Alcohol Naphthoic Acid Peptide Isothiocyanate Sodium Dodecyl Sarcosinate Sodium Citrate Sodium Acetate Sodium Dithiocarbamate Ethanol Ethanol Taqase
Centrifuge UV Spectrophotometer Agarose Gel Electrophoresis PCR Instrument
1. Collect about 5x107 cells and wash with cold PBS.
2. Add 400 ul RNA extraction solution (containing 6 mol/l peptide isothiocyanate, 0.5% sodium dodecyl sarcosinate, 0.025 mol/l sodium citrate pH 7.0, 0.25 mol/l sodium acetate pH 4.0, 0.5% dithiothreitol, 50% naphthoic acid) and mix well. 3.
3. Add 400 ul of chloroform, mix well, and centrifuge at 13 000 r/min for 15 min at 4℃. 4.
4. Take the supernatant and add an equal volume of isopropanol and leave it at 4°C for 30 min. 5.
5. Then centrifuge at 13 000 r/min for 15 min, aspirate the supernatant, wash the RNA spots with 75% ethanol once, dissolve in 100 ul of sterile double distilled water. 6.
6. Detect the purity of RNA by UV spectrophotometer ( A260/A280 should be 1.8~2.0) and prepare for use.
Reverse transcription and PCR amplification
1. Primers
2. 10 ul of total cellular RNA was added to 20 ul of AMV reverse transcriptase reaction system, and held at 42℃ for 30 min for reverse transcription to synthesize cDNA.
3. Then, the reverse transcription was terminated at 95℃ for 3 min.
4. Then, PCR was carried out under the action of Taq enzyme with mdr-1 and &beta ;2-MG primers on the target fragment of cDNA.
5. Pre-denaturation at 94°C for 2 min, followed by denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, and amplification for 35 cycles.
6. The final holding time was at 60°C for 7 min to ensure that the product was sufficiently extended.
7. 10 ul of amplification products were taken in 3% agarose gel electrophoresis, and then photographed under the UV lamp, and the allelic DNA band was considered as a positive control with mdr-1 cDNA, and equivocal DNA band was positive and vice versa is negative.