Total RNA extraction and reverse transcription

1) Materials

Reagents

Apparatus

TRIzol

Micro-centrifuge tube (1.5 mL)

Chloroform

Pipette (range: 1 μL-10 μL)

Isopropanol

Pipette (range: 0.1 mL-1 mL)

Ethanol (75%)

Centrifuge

5× HiScript III RT SuperMix

Spectrophotometer

DNase/RNase-free water

Thermostat

2) Procedure

Ø Total RNA extraction

Remove cell growth media.

Add 1 mL of TRIzol reagent directly to the cell culture plate to lyse the cells.

Pipet the lysate up and down several times to homogenize.

Incubate for 5 min to permit complete dissociation of the nucleoproteins complex.

Transfer the lysate to a tube.

Add 0.2 mL of chloroform, then securely cap the tube.

Upside down a few times, then incubate for 5 min at room temperature.

Centrifuge the sample for 15 min at 12,000 × g at 4℃.

Transfer the colorless upper aqueous phase to a new tube.

Add 0.5 mL of isopropanol, then incubate for 10 min at room temperature.

Centrifuge the sample for 10 min at 12,000 × g at 4℃.

Remove the the supernatant.

Add 1 mL of 75% Ethanol to the tube and pipet up and down several times.

Centrifuge the sample for 10 min at 12,000 × g at 4℃.

Remove the the supernatant and open the lid of tube.

Dry for few minutes at room temperature.

Dissolve RNA with 20 μL of DNase/RNase-free water.

Detect RNA concentration by using a spectrophotometer.

Ø Reverse transcription

Prepare reaction solution.

Reagents

Volume/mass

Total RNA

1 μg

5× HiScript III RT SuperMix

4 μL

DNase/RNase-free water

to 20 μL

Set up reaction program.

Step 1

37℃

15 min

Step 2

85℃

5 s

Step 3

10℃

Start the program.

 

Total RNA extraction and reverse transcription

1) Materials

Reagents

Apparatus

Template cDNA

PCR tube (0.2 mL)

Forward primer (20 μM) *

Pipette (range: 1 μL-10 μL)

Reverse primer (20 μM) *

Pipette (range: 1 μL-100 μL)

2× Rapid Taq Master Mix

Centrifuge

DNase/RNase-free water

Thermostat

2) Procedure

Ø Prepare reaction solution.

Reagents

Volume

Template cDNA

5 μL

Forward primer (20 μM) *

1 μL

Reverse primer (20 μM) *

1 μL

2× Rapid Taq Master Mix

25 μL

DNase/RNase-free water

to 50 μL

Ø Set up reaction program.

Step 1

98℃

10 s

Step 2

55℃

15 s

Step 3

72℃

60 s

Step 4

Goto step 1, 30 more times

Step 5

4℃

Ø Start the program.

* Sequence of primers

Gene

Forward primer

Reverse primer

OGN

GAATTCATGGAGACTGTGCACTCTA

CTCGAGTTAGAAGTATGACCCTA

 

Overlap extension PCR (OE PCR)

1) Materials

Reagents

Apparatus

ShRNA-1/-2/-N sequences

PCR tube (0.2 mL)

5× Annealing buffer for DNA oligos

Pipette (range: 1 μL-10 μL)

 

Centrifuge

 

Thermostat

2) Procedure

Ø Prepare reaction solution.

Reagents

Volume

ShRNA-1/-2/-N forward sequences (20 μM) *

10 μL

ShRNA-1/-2/-N reverse sequences (20 μM) *

10 μL

5× Annealing buffer for DNA oligos

5 μL

Ø Set up reaction program.

Step 1

95℃

2 min

Step 2

decrement 0.1℃ every 8 s

Step 3

Goto step 2, 700 more times

Step 4

4℃

Ø Start the program.

* Sequence of ShRNA

ShRNA

Forward

Reverse

ShRNA-1

5’-GATCCGATGCCCACATGC CTGTTGTCTGTGAAGCCACAGATGGGACA ACAGGCATGTGGGCATCTTTTTTG-3’

5’-GATCCGATGCCCACATGCCT GTTGTCTGTGAAGCCACAGATGGGACAAC AGGCATGTGGGCATCTTTTTTG-3’

ShRNA-2

5’-GATCCGTCAGCTTATCTTTATG CACCTGTGAAGCCACAGATGGGGTGCATAAAG ATAAGCTGACTTTTTTG-3’

5’-AATTCAAAAAAGTCAGCTT ATCTTTATGCACCCCATCTGTGGCTTCACAG GTGCATAAAGATAAGCTGACG -3’

ShRNA-N

5’-GATCCGCCAGA CCTGCAACCGATATCTGTGAAGCCACAGA TGGGATATCGGTTGCAGGTCTGGCTTTTTTG-3’

5’-AATTCAAAAAAGCCA GACCTGCAACCGATATCCCATCTGTGGCT TCACAGATATCGGTTGCAGGTCTGGCG -3’

 

Restriction enzyme double digestion

1) Materials

Reagents

Apparatus

Vector or OGN fragment

Micro-centrifuge tube (1.5 mL)

CutSmart

Pipette (range: 1 μL-10 μL)

EcoRI enzyme

Pipette (range: 1 μL-100 μL)

XhoI enzyme

Centrifuge

ddH2O

Thermostat

2) Procedure

Ø Prepare reaction solution in tubes.

Reagents

Volume/mass

Vector or OGN fragment

1 μg

CutSmart

5 μL

EcoRI enzyme

1 μL

XhoI enzyme

1 μL

ddH2O

to 50 μL

Ø Centrifuge the liquid to the bottom of the tubes.

Ø Place the tubes in the thermostat at 37℃ for 15 min and 65℃ for 20 min.

 

Ligation reaction

1) Materials

Reagents

Apparatus

Vector (digested pCMV-3Tag-1A)

Micro-centrifuge tube (1.5 mL)

OGN fragment

Pipette (range: 1 μL-10 μL)

T4 DNA Ligase

Pipette (range: 0.1 μL-2.5 μL)

10× T4 DNA Ligase Buffer

Centrifuge

ddH2O

Thermostat

2) Procedure

Ø Prepare reaction solution in a tube

Reagents

Volume/mass

Vector (digested pCMV-3Tag-1A)

0.03 pmol

OGN fragment

0.3 pmol

T4 DNA Ligase

1 μL

10× T4 DNA Ligase Buffer

1 μL

ddH2O

to 10 μL

Ø Centrifuge the liquid to the bottom of the tube.

Ø Place the tube in the thermostat at 16℃ overnight.

 

E. coli transformation

1) Materials

Reagents

Apparatus

pCMV-3Tag-1A-OGN

Pipette (range: 1 μL-10 μL)

DH5α chemically competent cell

Pipette (range: 1 μL-100 μL)

LB liquid medium (Kan-)

Petri dish

LB solid medium (Kan+)

Thermostat

 

Shaker

 

Incubator

2) Procedure

Ø Transformation

Thaw 30 μL of competent cells on ice for 1-2 min.

Mix the cells with 1 μL of plasmid (pCMV-3Tag-1A-OGN) in a tube.

Place the tube on ice for 30 min.

Heat shock: place the tube in the thermostat at 42℃ for 45 s.

Place the tube in ice bath immediately and cool it for 2-3 min.

Ø Resuscitation

Add 1 mL of LB liquid medium (Kan-) to the tube.

Mix the solution uniformly, and incubate it in the shaker at 37℃, 220 rpm for 45-60 min.

Ø Incubation

Aspirate 100 μL of transformed competent cells with one pipette.

Coat them uniformly on the LB solid media (Kan+).

Place the petri dish at room temperature until the solution is absorbed.

Turn the dish upside down and incubate the cells at 37℃ for 12-16 h.

 

Plasmid extraction

1) Materials

Reagents

Apparatus

TIANpure Midi Plasmid Kit

Micro-centrifuge tube (1.5 and 2 mL)

Bacterial solution

Pipette (range: 0.1 mL-1 mL)

PBS

Vortex machine

 

Centrifuge

2) Procedure

Ø Follow the manufacturer’s instruction.

 

Agarose gel electrophoresis

1) Materials

Reagents

Apparatus

Agarose

Conical flask (range: 250 mL)

TAE buffer

Pipette (range: 1 μL-10 μL)

YeaRed Nucleic Acid Gel Stain (10000×)

Electronic balance

DNA loading buffer (10×)

Spatula

DNA Marker

Microwave oven

DNA sample

Mold for creating gel

 

Electrophoresis chamber

 

UV transilluminator

 

Scalpel

2) Procedure

Ø Gel preparation

Place the weighing paper on the electronic balance, weigh 1g of agarose, and pour it into the conical flask.

Pour 100 mL of TAE buffer into the conical flask and shake it.

Cover the conical flask with the aluminum foil and place it into the microwave oven for 3 min, until the granules of agarose are well dissolved and invisible.

Use the pipette to transfer 10 μL of YeaRed nucleic acid gel stain into the conical flask and shake it.

Pour the gel into the mould and insert a “comb” into the gel.

Wait for 10 min until the gel is solidified.

Ø Electrophoresis

Pour about 400 mL of TAE buffer into the electrophoresis chamber.

Carefully take out the gel and place it in the electrophoresis chamber.

Mix the loading buffer and DNA sample by using the pipette.

Use the pipette to add 5 μL of marker in the first hole of the gel, then add 10 μL of each sample into the following holes.

Turn on the power of the electrophoresis chamber and wait for about 30 min (the specific time should depend on the loading buffer we used).

Ø Authentication and recycle

Take out the gel and place it on the UV tray. The UV tray will then be placed into the UV transilluminator.

Scan the gel.

Comparing with the marker reference, determine the target bands which will be extracted during the recycling process.

Use the scalpel to cut the target bands of the gel.

 

 

DNA Gel Extraction

1) Materials

Reagents

Apparatus

TIANgel Midi Purification Kit

Micro-centrifuge tube (1.5 mL)

Agarose gel

Pipette (range: 0.1 mL-1 mL)

 

Centrifuge

2) Procedure

Ø Follow the manufacturer’s instruction.

 

ATDC5 cells transformation

1) Materials

Reagents

Apparatus

pCMV-3Tag-1A-OGN

Tube (1.5 mL)

ATDC5 cells

Pipette (range: 1 μL-10 μL)

Lipofectamine 2000

Pipette (range: 1 μL-100 μL)

MEM medium

Pipette (range: 0.1 mL-1 mL)

OPTI-MEM medium

Incubator

2) Procedure

Ø Add 2 μg of pCMV-3Tag-1A-OGN, 2 μL of Lipofectamine 2000 into a tube with 200 μL of OPTI-MEM medium.

Ø Mix the solution gently and incubate at room temperature for 20 min.

Ø Replace the cell growth medium with 800 μL of MEM medium.

Ø Add the solution to the cell culture plate and shake gently.

Ø Incubate the cells at 37℃ for 4 h.

Ø Replace the MEM medium with the cell growth medium.

Ø Incubate the cells at 37℃ for another 24 h.

 

Alcian blue dyeing

1) Materials

Reagents

Apparatus

4% paraformaldehyde

Centrifuge tube (range: 15 mL)

Alcian blue 8GX

Electronic balance

Acetic acid

Pipette (range: 0.1 mL-1 mL)

PBS

Shaker

dH2O

Microscope

2) Procedure

Ø Prepare 1% Alcian blue dyeing solution.

Reagents

Volume/mass

Alcian blue 8GX

1 g

Acetic acid

3 mL

dH2O

97 mL

Ø Remove the cell growth medium and wash the cells with PBS.

Ø Fix the cells with 4% paraformaldehyde at room temperature for 15 min.

Ø Wash the cells with dH2O for three times (5 min each time).

Ø Incubate the cells with Alcian blue dyeing solution at 4℃ overnight.

Ø Recycle the dye solution.

Ø Wash the cells with dH2O for three times (5 min each time).

Ø The cells were maintained in dH2O and captured by using a microscope.

 

SDS-PAGE electrophoresis

1) Materials

Reagents

Apparatus

Protein sample

Pipette (range: 1 μL-10 μL)

Running buffer

Pipette (range: 0.1 μL-2.5 μL)

Loading buffer

Electrophoresis chamber

Marker

 

PAGE (12%)

 

2) Procedure

Ø Insert the prepared gel (12% BT) into the electrophoresis chamber.

Ø Add running buffer into the chamber until the gel is submerged.

Ø Mix the protein sample and loading buffer.

Ø Add 1.5 μL of marker to the first hole of the gel and add 10 μL of mixed sample solution to the following holes.

Ø Set up the electrophoresis program.

Step 1

80 V

30 min

Step 2

120 V

60 min

Ø Start the program.

 

Western blot

1) Materials

Reagents

Apparatus

Rabbit anti-OGN antibody

Trans-blot transfer

Mouse anti-β-actin antibody

NC membrane

HRP-conjugated Goat Anti-Rabbit IgG

Shaker

HRP-conjugated Goat Anti-Mouse IgG

Box (1 dm×1 dm×0.5 dm)

TBST solution

 

Clarity Western ECL Substrate

 

5% defatted milk solution

 

Gel with protein after SDS-PAGE

 

2) Procedure

Ø Transfer

Place the gel and NC membrane in the trans-blot transfer.

Submerge trans-blot transfer in the ice bath.

Set the transfer program at 320 mA, 90 min.

Start the program.

Ø Blocking

Turn the membrane to a box, add TBST solution into it, and rinse the membrane for 1-2 min.

Discard the TBST solution.

Add 5% defatted milk into the box until the membrane is submerged.

Place the box on the shaker at room temperature for 2 h.

Ø Primary antibody incubation

Discard the milk in the box.

Add rabbit anti-OGN antibody or mouse anti-β-actin antibody solution to the box.

Place the box on the shaker at 4℃ overnight.

Recycle the primary antibody solution.

Rinse the membrane with TBST solution three times, 15 min for each time.

Ø Secondary antibody incubation

Discard the TBST solution in the box.

Add HRP-conjugated Goat Anti-Rabbit IgG or HRP-conjugated Goat Anti-Mouse IgG solution to the box.

Place the box on the shaker at room temperature for 2 h.

Recycle the secondary antibody solution.

Rinse the membrane with TBST solution three times, 15 min for each time.

Ø Detection of proteins

Add Clarity Western ECL Substrate solution into the box until the membrane is evenly soaked.

Use the software GelCap to scan the membrane.

 

 

 

Quantitative Real-Time PCR (qRT-PCR)

1) Materials

Reagents

Apparatus

ChamQ SYBR qPCR Master Mix (2×)

PCR tubes (0.1 mL)

Template cDNA

Pipette (range: 1 μL-10 μL)

Forward primer (20 μM) *

Pipette (range: 0.1 μL-2.5 μL)

Reverse primer (20 μM) *

qPCR thermal cycler

ddH2O

 

2) Procedure

Ø Prepare reaction solution.

Reagents

Volume

ChamQ SYBR qPCR Master Mix (2×)

10 μL

Template cDNA

0.1 μL

Forward primer (20 μM) *

0.25 μL

Reverse primer (20 μM) *

0.25 μL

ddH2O

9.5 μL

Ø Set up the reaction program.

Step 1

95℃

10 s

Step 2

95℃

5 s

Step 3

56℃

20 s

Step 4

72

10 s

Step 5

Goto step 2, 45 more times

Step 6

72℃

10 min

Step 7

Melt Curve 50 to 95℃, increment 0.5℃ for 5 s

Step 8

10℃

5 min

End

Ø Start the program.

* Sequence of primers

Gene

Forward primer

Reverse primer

OGN

ACCATAACGACCTGGAATCTGT

AACGAGTGTCATTAGCCTTGC

Col2α1

GGGAATGTCCTCTGCGATGAC

GAAGGGGATCTCGGGGTTG

Aggrecan

CCTGCTACTTCATCGACCCC

AGATGCTGTTGACTCGAACCT

Sox9

GAGCCGGATCTGAAGAGGGA

GCTTGACGTGTGGCTTGTTC

MMP3

ACATGGAGACTTTGTCCCTTTTG

TTGGCTGAGTGGTAGAGTCCC

MMP13

CTTCTTCTTGTTGAGCTGGACTC

CTGTGGAGGTCACTGTAGACT

β-Actin

GGCTGTATTCCCCTCCATCG

CCAGTTGGTAACAATGCCATGT