Team:CSU CHINA/Protocols

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PROTOCOLS

PROTOCALS

I.Endogenous transcription factor(TF) selection （Bioinformatics）

  1. screen for highly expressed genes in tumor groups in all invasive breast cancer from GEPIA database. Then, 245 genes is selected initially.
  2. 86 normal breast transcriptome samples and 303 tumor transcriptome samples were downloaded from TCGA (The Cancer Genome Atlas) database. The differentially expressed genes were screened by EdgeR algorithm. And all genes were selected in the GEPIA results set (245).
  3. Using the GEPIA database to test the most specific differential gene in invasive breast cancer, and finding out that 11 overlaps with the previously selected 245 in PART 2.
  4. Set priority order for transcription factor identification. Finally, 9 transcription factors were identified

II.mRNA qPCR

1.Reverse Transcription for mRNA

Step One 12μl Water 10μl
RNA 1mg
Random Primer 1μl
Operate the PCR instrument at 65℃ for 5min and put on ice immediately after taking it out
Step Two 8μl 5X Reaction Buffer 4μl
Rib RNase Inhibitor 1μl
10Mμ dNTP mix 2μl
Reversed Aid RT 1μl
Total:20μl

Then operate the PCR instrument：25℃ 5min→42℃ 60min→70℃ 5min→4℃ ∞

2.qPCR of cDNA obtained from miRNA RT reaction

SYBR 5μl
F Primer 0.2μl
R Primer 0.2μl
ddH2O 2.6μl
cDNA 2μl
Total 10μl
Then put them for qPCR machine
95℃ for 10min
95℃ for 15s 40 cycles
60℃ for 60s
4℃ reserved

III.miRNA series methods

1.RT Reaction for microRNA

1.1 Preparing for RT Reaction Mix

In a 1.5ml microcentrifuge tube , prepare RT Reaction Mix according to the following table:

Component Volume (1 reaction) Volume (10 reactions)
100mM dNTPs (with dTTP) 0.15μl 1.65μl
MultiScribeTM Rerverse Transcriptase , 50U/μl 1.00μl 11.00μl
10X Reverse Transcription Buffer 1.50μl 16.50μl
RNase Inhibitor , 20U/μl 1.50μl 16.50μl
Nuclease-free Water 4.16μl 45.76μl
Total RT Reaction Mix volume 7.00μl 77.00μl

Place the RT Reaction Mix on ice, then immediately proceed as followed procedures

Primer(RT) design：stem loop method

1.2 Performing reverse transcription

  1. Combine 3μl of 5X RT Primer and 5 μl of double-stranded template in a reaction tube.
  2. Mix thoroughly, then centrifuge briefly to collect the contents at the bottom of the tube.
  3. Incubate at 85℃ for 5min
  4. Incubate at 60℃ for 5min, then place on ice
  5. Add 7μl of RT Reaction Mix to each reaction tube. (details from “Preparing for RT Reaction Mix” showed above)
  6. Centrifuge briefly to collect the contents at the bottom of the tubes

Place on ice and proceed immediately to “Performing reverse transcription” below.

1.3 Performing reverse transcription

Place the reaction tubes into a thermal cycler, then incubate using standard cycling, a reaction volume of 15.0μl, and the following settings.

Step Temperature Time
Reverse transcription 16℃ 30min
42℃ 30min
Stop reaction 85℃ 5min
Hold 4℃ Hold

The RT reaction products are stored at -25℃

2.qPCR of cDNA obtained from microRNA reverse transcription

SYBR 5μl
F Primer 0.2μl
R Primer 0.2μl
ddH2O 2.6μl
cDNA 2μl
Total 10μl
Then put them for qPCR machine
95℃ for 10min
95℃ for 15s 40 cycles
60℃ for 60s
4℃ reserved

IV.Sponge

1.PCR for Sponge

Reagent Volume(μl)
5X Buffer 10
DMSO 4
dNTP 4
Forward Primer 2
Reverse Primer 2
Targeted DNA fragment 1
phusion 1
ddH2O 26
In Total 50

Then put the reaction system to the PCR machine

Cycle Temperature Time
94℃ 5min
10 cycles Add 0.5℃ after per cycle 94℃ 30s
55℃ 30s
72℃ 30s
25 cycles 94℃ 30s
60℃ 30s
72℃ 30s
72℃ 7min
4℃ reserved

V.miRNA-BS

1.PCR for Binding site（BS）

94℃ 5min
10 cycles Add 0.5℃ after per cycle 94℃ 30s
55℃ 30s
72℃ 30s
25 cycles 94℃ 30s
60℃ 30s
72℃ 30s
72℃ 7min
4℃ reserved

2.Digestion of Binding site（BS）

BS-1 20μl
BS-2 20μl
Bbs1 2μl
10XBuffer 5μl
ddH2O 6μl
Total 50μl

Examples:

BS-1: miR141-BS

BS-2: miR148-b-BS

VI.Double digestion to construct plasmid

1.Digestion

DNA Vector
In Total 10 15
Kpn1 1 1
Xho1 1 1
10XBuffer 2 2
ddH2O 6 11

2.Connection

Fragment 3μl
Vector 10μl
10XBuffer 2μl
T4 DNA Ligase 3μl
ddH2O 2μl
Total 20μl

VII.Golden Gate methods

1.Goldengate splicing

Example that synthase Module2: making s(ESR1)p and Sponge connected

Vector 2μl
Sponge 1μl
s(ESR1)p 1μl
10XT4 Ligase Buffer 2μl
10XBuffer 2μl
Esp3L 1μl
ddH2O 11μl
Total 20μl

2.PCR amplification

Step 1(X10)
37℃ 5min
16℃ 10min
Step 2
37℃ 15min
50℃ 5min
80℃ 5min
Step 3
16℃ reserved

Then the 10mM ligation was chemically transformed, screened and cloned by blue and white method, and the positive clones were made into colony PCR and sent for sequencing

VIII.Luciferase assay

1.Measurement of Luciferase

  1. discard the medium with Luciferase plasmid 48h after transfection and wash it with PBS
  2. add 1XLys Buffer 50 mu/hole (diluted from 5XLys Buffer)
  3. freeze at -80℃ for 30 minutes
  4. take out, natural melting on ice, centrifuge set 4℃ ready
  5. take out the precooled ep tube and scrape the cells with tip head to transfer the cells into the ep tube
  6. 13,000g centrifuge for 10min, set aside

Detection: take a new ep tube, add 20 microns luciferase detection reagent (stored at -80℃), add 10 microns to each tube to get the supernatant, mix evenly, and test under the enzyme label meter.

2.Measurement of LacZ

  1. discard the medium with Luciferase plasmid 48h after transfection and wash it with PBS
  2. add 1XLys Buffer 50 mu/hole (diluted from 5XLys Buffer)
  3. freeze at -80℃ for 30 minutes
  4. take out, natural melting on ice, centrifuge set 4℃ ready
  5. take out the precooled ep tube and scrape the cells with tip head to transfer the cells into the ep tube
  6. 13,000g centrifuge for 10min, set aside

Detection:

a. Take 96-well plates and add 100 micron LacZ Buffer into each hole
b. The supernatant obtained by adding 10 microns per hole
c. The temperature of 37 ℃) for 30 min
d. When the sample turned yellow, 50 micron l 1 micron NaCO*10HO was added to terminate the reaction232
e. LacZ was detected with microplate reader, wavelength =405nm

IX.Cell Biological protocols

1.Cell planking

  1. the medium
  2. wash it with PBS (1~2ml)
  3. trypsin (1ml) can be digested in the 40s to 1990s, during which time it can be placed in the incubator
  4. add 5ml medium to terminate digestion
  5. transfer into a 15ml tube, suck out 3ml for transmission, and dilute the remaining 3ml to 6ml with pechil
  6. add the diluted pge to a 24-well plate at 500 microns per hole, and then test luciferase

X.Molecular Colon basic protocols

1.PCR Reaction

Reagent Volume(μl)
5X Buffer 10
DMSO 4
dNTP 4
Forward Primer 2
Reverse Primer 2
Targeted DNA fragment 1
phusion 1
ddH2O 26
In Total 50

2.PCR production purification

——QIAquickR PCR Purification Kit

  1. PB Buffer was added to the PCR product at a volume of 5:1
  2. The sample was added into the column, centrifuged for 30-60s, and the supernatant was discarded after centrifugation
  3. Add 750 micron Buffer PE column, centrifuge for 30-60s and discard supernatant
  4. Then centrifuge for 1min and discard the residual liquid
  5. Insert the column into the ep tube
  6. Add 50 micron PB Buffer(10m micron Tris*HCL,pH8.5) or HO(ph 7.0-8.5) to the center of the membrane and centrifuge for 1min.2Elution with 30 microns will increase the concentration and centrifuge after 1min

3.Gel recycling

——QIAquickR PCR Purification Kit

  1. a blade to remove the desired fragment from the agarose gel
  2. Weigh the Gel in a colorless tube and add triploid Buffer OG to double the size of the Gel (100mg Gel=100 microns).The filtration column can hold up to 400mg (agarose > 2% gel, plus 6 times the volume of Buffer OG).
  3. Heat it at 50℃ for 10min (until the glue melts), and turn on the vortex every 2-3min to promote the glue melt.(if the liquid is orange or purple at this time, add 10 mil 3 sodium acetate, pH5.0 and mix until the mixture turns yellow.)If the tube is yellow, it is normal.
  4. Add Isopropanol with the same volume of glue and mix
  5. Spon column was placed on the 2ml collection tube, centrifuged for 1min to discard the supernatant, and the operation was repeated if the volume was > 800 microns.
  6. If the DNA is eventually used for sequencing, in vitro transcription, or microinjection, 500 mul Buffer OG is added to the column, centrifuged, and supernatant is discarded
  7. Add 600 mu Buffer PE, centrifuge for 1min, discard supernatant and repeat operation.(if the DNA is used in salt-sensitive experiments, sequencing and connection should be carried out. After adding Buffer PE, the tube should be left for 2-5min and centrifuged for 1min to discard the supernatant.)
  8. Place the column in a 1.5ml ep tube
  9. Add 50 micron Buffer EB (10 micron tris-hcl, pH8.5) or water to the center of QIAquick membrane and centrifuge for 1min.In order to obtain a high concentration of DNA, a 30mul Buffer EB can be used and left for 1-4min before centrifugation for 1min

4.Extraction of Plasmid （Mini）

a small amount of silicon matrix column suitable for the extraction of plasmid DNA up to 35 microns from 1-5ml bacterial culture solution

Remove 15ml LB liquid medium and divide into 4 tubes

  1. Strains were inoculated in 15ml LB+ antibiotic culture tube and cultured in a shaking table at 37℃ for 12~16h
  2. Centrifuge 10,000g for 1min to collect the bacteria
  3. Pour and discard the culture in 15ml culture, tap and absorb the residual liquid on the absorbent paper, then mix with 750l Buffer P1/RNase A, and suspend the bacteria in high-speed vortex (until no more bacterial clumps are seen).
  4. Add 750 micron Buffer P2 to the resuspension and mix it upside down for 8 to 10 times (no vortex, when the solution becomes thick and transparent, it indicates that the bacteria has fully cracked).
  5. +1.4ml Buffer P3, immediately reverse 8-10 times to neutralize
  6. 13,000g centrifugation for 10min
  7. HiPure DNA Mini Column II was loaded into the collection tube, the supernatant was transferred to the adsorption Column, and centrifuged at 133g for 1min
  8. Pour the filtrate into the adsorption column with +500 micron Buffer PW1 and centrifuge at 13,000g for 30-60s (it can be omitted, but it cannot be omitted when treating strains containing nuclease, such as HB10).
  9. Pour filtrate, +600 microns PW2,13,000g centrifuge 30-60s
  10. Repeat step 9
  11. Pour filtrate and centrifuge for 1min at 133g
  12. Transfer the adsorption column to a 1.5ml new EP tube, add 30~100 micron Elusion Buffer, leave for 1min, and centrifuge for 1min at 13,000g
  13. Discard column and store at -20℃

5.Preparation of competent cells

- DH5a CELL

  1. Put the cells on ice and let stand for 20min, then precool the Ep tube
  2. The 2 mul plasmid was added to the cell and placed on the ice for 20min
  3. Heat shock 42℃, 1990s, followed by 5min on ice
  4. Add 500 microns of antibiotic-free liquid medium and incubate in a shaking table at 37℃ for 30-60min
  5. Add antibiotics (1:1000) 50-100ug/ml to 15 mul tube

6.Transfection

For one example：

PGL4.22-Promoter 0ng 5 ng 10 ng 20 ng
β-Gal(line lacZ) 10ng 10 ng 10 ng 10 ng
Empty 90 ng 85 ng 80 ng 70 ng
The total plasmid DNA content of each hole (12 holes in total) in the 24-well plate was 100ng.According to the Lipomax: DNA = 3 mu l;The 100ng ratio is configured with the following optin mixture
+25μl Optin-DNA Incubate 5min
+25μl Optin-Lipomax incubate5min
Blend, then incubate for 20min（the system has a total of 50μl）

7.Extraction of RNA

For adherent cells:

  1. Remove the medium and wash once with 1-2ml cold PBS
  2. Remove PBS and lyse cells directly, adding 1ml Trizol per 100mm dish
  3. The pipette blows the lysate several times and suspenders it
  4. Incubate homogenate for 5min
  5. Add 0.2ml chloroform /1ml Trizol, shake tube for 15s, room temperature 2~3min
  6. The mixture was centrifuged at 2~8℃ at 12,000g for 15min, and the mixture was stratified. The lower layer was phenolic or chloroform, and the middle and upper layers were aqueous phases
  7. Transfer the water phase to the new ep tube, about 500 microns
  8. Add 0.5ml isopropanol /1ml Trizol, mix them upside down and incubate for 10min
  9. RNA precipitation was collected after centrifugation at 2~8℃ for 10min at no more than 12,000g
  10. Discard the supernatant and absorb the residual liquid
  11. Wash RNA with 75% ethanol once, add at least 1ml 75% ethanol, centrifuge at 2~8℃ for 5min (no more than 7,500g)
  12. Dissolve RNA in 20 micron RNase free water, blow the solution several times, and incubate the solution at 55~60℃ for 10min

XI.DAPI staining

For Anchorage-dependent cells like MBA-MD-231 and HBL-100

  1. Wash the media with PBS for three times
  2. Incubate with PFA for 30-60 minutes
  3. Wash the media with PBS for three times
  4. Incubate in a solution of 1µg/ml 4,6-diamidino-2-phenylindole (DAPI) for 30 minutes
  5. Count cells under fluorescence microscope
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