Total RNA Kit : Omega: (Cat.No.R6834)
First Strand cDNA Synthesis Kit: GeneCopoeia (Cat. No. C0210A)
2xAllinOneTMQ-PCRMix: GeneCopoeia (cat.No.D0101A)
Primer synthesis: invitrogen
experiment apparatusReal Time PCR: ABI
Experimental MaterialsThe sample is a cell culture solution of 5 human origins, and their numbers are: NO.I, No.2, No.3, No.4, No.5, wherein No.1 is a control group, and the other four are different samples. group.
Experimental procedureRNA extraction
1) Sample processing: Add an appropriate amount of sample to 1 ml of TRK lysate homogenate sample. The undissolved impurities were removed by centrifugation at 14,000 × g for 5 min at room temperature, and the supernatant was carefully transferred to a 1.5 ml centrifuge tube.
2) Add an equal volume of 70% ethanol to the lysate, mix by whipping or vortexing.
3) Place the column in the collection tube and transfer the mixture to the column. Centrifuge at 10,000 × g for 30-60 seconds at room temperature and discard the filtrate.
4) Place the column in a new collection tube, add 300ul of RNA Wash Buffer I to the column, centrifuge according to the above conditions, and discard the filtrate. Put the column sleeve back into the collection tube.
5) DNase digestion: Prepare DNASE Digestion Buffer (Digestion Buffer, 73.5ul; RNase-Free DNase I, 1.5ul), mix well, transfer the digestive juice to the center of the column membrane and let stand for 15 minutes at room temperature.
6) Add 500ul RNA Wash Buffer I to the column, centrifuge according to the above conditions, and discard the filtrate.
7) Put the column sleeve back into the collection tube, add 500ul RNA Wash Buffer II to the column, centrifuge according to the above conditions, and discard the filtrate.
8) Put the column sleeve back into the new collection tube, add 500ul RNA Wash Buffer II to the column, centrifuge according to the above conditions, and discard the filtrate.
9) Put the column sleeve back into the collection tube and centrifuge the empty column for 10,000 xg for 2 min to dry the column matrix.
10) Place the column on a 1.5 ml centrifuge tube, add 30-100 ul of DEPC Water column substrate, and let stand for 2 min at room temperature. RNA was eluted by centrifugation at 10,000 xg for 1 min.
11) Determination of RNA concentration
The lul extracted RNA was diluted 10 times with DEPC water. RNA concentration was determined on Nanodrop, and DEPC water was used as a blank control, and RNA concentration and OD260/OD280 were recorded.
2. RNA electrophoresis detection
1) Preparation of denatured rubber
Take 1g of Agarose 75ml deionized water and boil. Cool to about 70 °C and add 10ml of 10×Mops and 15ml of formaldehyde and EB. Pour the glue onto the rubber plate of the wide-mouth comb and close the lid.
2) Preparation of electrophoresis buffer (1×Mops)
Take 50ml 10×Mops. Dilute to 500 ml with deionized water, pour into the electrophoresis tank, and add EB to the running buffer.
3) RNA sample processing
Take the RNA sample 3u1.10×Mops 2u1. Finally, add DEPC water to 20ul, and then cool at 65 °C for 10min. Electrophoresis can be performed by adding 2ul of 10×RNA loadingbuffer.
4) RNA electric ice
The RNA gel was placed in an electrophoresis tank, and electrophoresed at 100V for about 5 minutes. Then, the treated RNA sample was spotted in the spotted well, and electrophoresed to about 2/3 of the bromophenol blue to the gel at about 100V.
3. Reverse transcription
1) Dissolve the reagents required for the reaction, mix it upside down slightly, centrifuge briefly, and place on ice for later use.
2) Preparation of RNA-Primer Mix (all reaction solutions were prepared on ice) The following reagents were added to a pre-cooled RNase free reaction tube to a total volume of 13 μl.
Reagent component volume final concentration
Total RNA 1μg
60μM Oligo(dT)18 1μL 2.4μM
DEPC water to a total volume of 13μl
3) RNA denaturation
The RNA-Primer Mix was mixed, briefly centrifuged, and denatured at 65 ° C for 10 min and placed on ice immediately.
4) Preparation of reverse transcription reaction solution
The following reagents were added to the RNA-Primer Mix reaction tube to a total volume of 25 μl.
Reagent component volume final concentration
RNA-Primer Mix 13μl
5×RT Reaction Buffer 5μl 1×
25mM dNTP 1μl 1mM
25U/μl RNase Inhibitor 1μl 1U/μl
200U/μl M-MLV RTase 1μl 8U/μl
DEPC water 4μl
Total volume 25μl
5) Reverse transcription reaction
Mix the reaction Mix and incubate for 1 h at 42 ° C after brief centrifugation.
6) Inactivate and preserve the reverse transcript
After the end of the reaction, the inactivation treatment was carried out at 85 ° C for 5 min, and finally the reverse transcription product was stored at -20 ° C.
4. Quantitative PCR experiments
The relative quantitative analysis was carried out by the dye method (SYBR Green I), and the experimental design was designed according to the ΔΔCt analytical method. The experimental steps are as follows:
1) Melt 2XAllinOneTMQ-PCR Mix at room temperature. Gently mix upside down and briefly centrifuge. At the same time, always keep away from light during use.
2) Preparation of PCR reaction mix (operating on ice)
Reagent dosage final concentration
2XAllinOneTMQ-PCR Mix 10μl 1×
ddH2O 1μl
PCR Forward Primer (4μM) 2μl 0.4μM
PCR Reverse Primer (4μM) 2μl 0.4μM
cDNA 5μl
Total volume 20μl
Note: NTC (No Template Control) was also designed in the experiment, which is a negative control, that is, water is used instead of template cDNA in the reaction, and other reagents are unchanged. Therefore, the quality control system is polluted. The PCR reaction mix was quickly mixed and added to an 8-tube tube.
3) Centrifuge the 8 tubes briefly to ensure that all the reaction solution is at the bottom of the reaction well.
4) The PCR reaction was carried out using a standard three-step procedure:
Number of cycles Step Temperature Time Detection
1 Pre-denaturation 95°C 10min Off
40 Denaturation 95°C 10sec Off
Annealed 57°C 20sec Off
Extend 72°C 15sec On
5) After the PCR reaction. Use the following procedure for melting curve analysis
Temperature temperature interval time detection
72°C~95°C 0.5°C 6sec/each On
30°C 30sec off
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