Taqman Results and Transfection

20/01/2010 -

The Taqman results gave a circumstantial relationship between miR-34 and Zmpste24, as it was seen in cells high in miR-34 there was a low level of Zmpste24 and vice versa, indicating that Zmpste24 was a likely target for miR-34.

10 more cultures were mini-prepped and digested using the Sac1 restriction enzyme. The gel image again revealed the insert had been incorporated in the wrong way. But every vector had the insert, which is unusual due to the low efficiency rate of the transformation so it is thought that the undigested vector is predominant on the plate.

A transfection was set up using the SW1353 cells which had been plated and were allowed to grow until they were confluent. The cells were tested with a control (scrambled) which scrambles the miR-34 binding site and a miR-34 mimic at 3 different concentrations, 10nM, 20nM, and 50nM.

The following volumes were added to 200 microL serum free medium in separate eppendorfs

Control:

10nM - 0.4 microL
20nM - 0.8 microL
50nM - 2 microL

miR-34 mimic

10nM - 1 microL
20nM - 2 microL
50nM - 5 microL

The eppendorfs were then incubated at room temperature for 5 minutes.

During this incubation period a 15x mastermix of lipofectamine 2000 (a transfection agent which, when combined with the mimic, will transport the mimic into the cell) and serum free medium was made.

1X = 4 microL lipofectamine, 196 microL serum free medium
15X = 60 microL lipofectamine, 2.9mls serum free medium

Once the incubation period was over 200 microL of the mastermix was added to each eppendorf. These were then incubated at room temperature for 20 minutes.

After this incubation period was finished the solutions were added to each well in a drop-wise fashion. The plate was then incubated at 37 degrees Celsius for 48 hours. How the 6 well plate was organised is shown below.



21/01/2010 -

A PCR purification was done using a QIAgen QIAquick PCR purification as per the protocol provided with the following changes:

100microL PB1 added to sample
50microL water used to elute DNA rather than EB

The DNA was then digested using 10microL H3 enzyme, 10microL H3 buffer, 30microL water (this brings the final volume to 100microL). The sample was then digested for 3 hours at 37 degrees Celsius.

5 microL of the sample was run on a 1.5% agarose gel to test the digestion. The digestion worked very well and the insert and vector were cleanly cut. Again the insert was 400bp, but this time no background vector appeared on the gel image.

25/01/2010

A western blot was set up using the transfected cells. The total protein concentration was measured and the following volumes were loaded (+4 microL loading buffer) to ensure the same amount of protein was loaded in each lane (80nM)

Transfection Absorbance Protein Conc. (ng/microL) Volume loaded (microL)
Scrambled10 0.48 4.8 16.6
Scrambled 20 0.65 6.4 12.5
Scrambled 50 0.57 5.7 14
miR-34 10 0.63 6.3 12.7
miR-34 20 0.51 5.1 9.8
miR-34 50 0.52 5.2 5.1

The protein and loading buffer were boiled for 5 minutes at 90 degrees Celsius on a hot block before loading. The gel was then run at 150V for 1 hour.

Once the gel has finished running it was transferred to a membrane using the same method as previously described.

While the proteins were being transferred to the membrane 12 more colonies were picked and incubated in LB + 5 microL ampicillin and incubated overnight, shaking, at 37 degrees Celsius.

26/01/2010

The 'quick and dirty' mini-prep method was used to purify the plasmid DNA.

The primary Zmpste24 antibody was applied to the membrane yesterday and it was left in the cold room overnight.

The secondary antibody was then applied and the membrane put on the shaking platform for 1 hour.

After this hour it was washed in PBS tween, 2 x 5 mins on the shaking platform. Then 1ml of a chemiluminescent was added and left to incubate for 1 minute. The membrane was then transferred to an AUTO-RAD sheet and left to develop for 1, 5 and 10 minutes.
The developed AUTO-RAD sheets can be seen in my lab diary.

The plasmid DNA was digested with Sac1 as previously and the samples run on an electrophoresis gel. Only one vector had incorporated the insert, but again in the wrong orientation.

28/1/2010

A flask of P.34 cells were harvested and protein and RNA was extracted using the same method as previously described. New medium was added to the second flask to keep the cells alive until we can photograph them. These cells are now reaching the stage of senescence.

Taqman MicroRNA Assay

19/01/2010 -

A Taqman microRNA assay was set up using the cDNA created yesterday. I started with 4 different types of cDNA :

1 - HeLa + random hexamers
2 - HeLa + miR-34 primers
3 - SW1353 + random hexamers
4 - SW1353 + miR-34 primers

Each of these cDNAs were then diluted 1 in 100 (2 µl cDNA in 198 µl water)

Then using the undiluted cDNA a number of dilutions were set up:
For random hexamers (use either HeLa or SW1353 cDNA)
1/10 - 2 µl undiluted cDNA + 18 µl water
1/100 - 2 µl cDNA + 198 µl water
1/1000 - 54 µl water + 6 µl 1/100 dilution
1/10000 - 54 µl water + 6 µl 1/1000 dilution.

Vortex briefly after each dilution.

For miR-34 (use either SW1353 or HeLa cDNA)
1/10 - 2 µl cDNA + 18 µl water
1/100 - 2 µl cDNA in 198 µl water

These dilutions were then used to create 18S, Zmpste24 and miR-34 standard curves as follows:

18S standard curve:
6 eppendorfs were set up as follows



Sample gene -
This method is the same for both Zmpste24 and miR-34 standard curves. For Zmpste24 use random hexamer dilutions and for miR-34 use the miR-34 primer dilutions



Once each of the standard curves have been prepared the Taqman plate was then loaded as follows: -

A1 = 2µl HeLa RH + 8µl water
A2 = 2µl SW1353 RH + 8µl water
B1 = 10µl HeLa RH
B2 = 10µl SW1353 RH
C1 = 10µl HeLa miR-34 primers
C2 = 10µl SW1353 miR-34 primers
A3-A8 = 10µl 18S standard curve
B3-B8 = 10µl Zmpste24 standard curve
C3-C8 = 10µl miR-34 standard curve
A12, B12, C12 = 10µl water (which acts as a negative control)

10x mastermix was made up as follows:


15µl of the appropriate mix was added to the appropriate well:
A1-A8, A12 = 15µl 18S
B1-B8, B12 = 15µl Zmpste24
C1-C8, C12 = 15µl miR-34

The plate was then run in the Taqman machine.

Transformations, Digestions and Reverse Transcriptions.

12/01/2010 -

Using the ligations Tracey created over the Christmas holidays further transformations were set up using the DHS-Alpha bacteria, following the same protocol as previously. The agar plates were then incubated overnight at 37 degrees Celsius.

13/01/2010 -

Lots of colonies grew on the 1 µl and 3 µl plates but not as many on the 5 µl plate. It was decided to pick colonies from the 1 µl and 3 µl plates but not the 5 µl plates as these colonies did not work as well when Tracey did the transformation, but they will be left as a back up. 5 colonies were picked from each plate. These were then incubated in LB + 5 µl ampicillin overnight at 37 degrees Celsius, shaking overnight.

14/01/2010 -

DNA purification using a quick method which does not use columns.
This method is used when there are a lot of samples (10 in my case) as the QIAgen mini-prep method is expensice, by not using columns it makes it cheaper. The protocol is as follows:
1) Take 1.5ml culture and spin at 13,000rpm for 2 minutes to form a pellet.
2) Remove supernatant
3) Add 100 µl of P1 resuspension buffer
4) Resuspend pellet very gently.
5) Add 200 µl of P2 buffer
6) Add 150 µl of P3 buffer
7) Invert 6 times
8) Centrifuge at 13,000rpm for 3 minutes
9) Add supernatant to 1ml of 100% ethanol (in clean eppendorf tubes)
10) Vortex
11) Centrifuge at 13,000 rpm for 10 minutes
12) Remove ethanol, use a smaller pipette to remove dregs
13) Leave to air dry
14) Resuspend pellet in 40 µl water

Then two digests were set up using H3 and Sac1 restriction enzymes.

1 µl DNA
1 µl enzyme buffer
1 µl enzyme
7 µl water

These digestions were incubated at 37 degrees Celsius for 1 hour. Once the digestion was completed the samples were run on an agarose electrophoresis gel. The image revealed none of the vectors had taken up the insert.

15/01/2010 - 17/01/2010 -

Over the weekend Tracey set up a number of mini-preps using the colonies I had grown previously. These were purified using the no columns method then digested using Sac1. After running on an electrophoresis gel it was revealed no insert had been taken up.

Tracey then re-ligated the vectors which had contained the insert in the wrong orientation. The DNA was purified and digested with Sac1. Again it was seen that insert has been incorporated, but in the wrong orientation. This may have been because not all the initial vector had been re-ligated and this was what was coming through on the gel.

18/01/2010 -

Reverse Transcription - creation of cDNA for use in a Taqman MicroRNA assay.

Tracey had previously extracted RNA from SW1353 and HeLa cells, both of which are Cancer cell lines (SW1353= Human Bone Chondrosarcoma, HeLa= Cervical Carcinoma)

This RNA was very concentrated so was diluted 1 in 5 (5 µl RNA in 25 µl water). The nanodrop was then used to check the diluted concetration:

SW1353 = 711.8 ng/ µl
HeLa = 6.18 ng/ µl

This was still very concentrated so the RNA was diluted again

SW1353 = 1.4 µl in 7.6 µl water
HeLa = 1.6 µl in 7.4 µl water

This gave a final volume of 9 µl

For each cell line 2 tubes were set up one which will use random hexamers and one which would use miR-34 primers.

To the diluted RNA 2 µl of either random hexamers or miR-34 primers was added. The tubes were then incubated at 70 degrees Celsius for 10 minutes, using a hot block.

While the tubes were incubating a 5x mastermix of reverse transcription agents was made up. By making this mastermix it helps with precision of pipetting and helps to keep the volume of each agent pipetted in each tube constant, helping with accuracy.

On ice add : -

1x 5x
4 µl 5 x 1st strand buffer 20 µl
2 µl DTT 10 µl
1 µl dNTPs 5 µl
1 µl Superscript RT 5 µl
1 µl RNase Inhibitor 5 µl

9 µl of the mastermix was added to each tube
These tubes were then incubated at 42 degrees Celsius for 1 hour then at 70 degrees Celsius for 10 minutes
The tubes were then stored at -20 degrees Celsius.

Tracey picked 12 colonies and incubated then in LB + 5 µl ampicillin overnight at 37 degrees Celsius, shaking, overnight.

Christmas Holidays

Over the Christmas holidays Tracey set up a number of ligations with ratios of 1 µl, 3 µl and 5 µl insert to 1 µl of vector. From these ligations transformations were done which were consequently plated onto agar. Once these colonies had grown 10 were picked and Ph-Chl cleaned and digested with H3 (the restriction enzyme HindIII). These samples were then run on an agarose electrophoresis gel to check if the insert had been incorporated into the vector. The gel image revealed of the 10 colonies only 1 had taken up the vector.

Next it needed to be checked whether the insert had been incorporated into the vector in the correct orientation. This is done by digesting with the restriction enzyme Sac1. The vector and insert both contain Sac1 restriction sites, the site with in the insert is off centre allowing for two different sized fragments depending on the orientation of the insert. After digestion with Sac1 if the insert is in the correct orientation the fragment is 100bp, if it is the wrong way round the fragment will be 400bp.

Once the digestion of Sac1 was complete the fragments were run on an electrophoresis gel. The gel image revealed the insert was 400bp and therefore the insert was in the incorrect orientation.

These vectors will still be used as positive controls as they contain the anti-sense strand for the miR-34 binding site. The fact that any insert was incorporated was also a good thing as it illustrated that the transformation has actually been working. It also indicates a very low efficiency for the experiment.

Taqman results, Plasmid DNA purification and re-transformation

2/12/2009 -

The Taqman assay showed each of the cell lines expressed Zmpste24 at differing levels. The assay worked well which is encouraging as we were unsure that the RNA was of a good quality.

The results were analysed using Excel. All cycles in 18S should be amplifying within 1.5 cts (cycle thresholds) of each other, any that were not were removed from the analysis. These cell lines were unlikely to have RNA that was of sufficient quality. Cycles of Zmpste24 should not be amplifying at the same time as we want them to be different.

A western blot was run using antibodies specific for GAPDH, a protein found in all cells. This is run as a control to make sure the same amount of protein was run in each lane. The antibodies were annealed to the membrane and proteins in the same way as before. Then 9ml of water and 1 ml of chemiluminescent was poured onto the membrane and left for 1 minute. The membrane was then put into a light-proof case and exposed with an AUTO-RAD sheet for 1 minute then developed. The resulting picture showed that there was the same amount of P.29 and P.30 protein loaded in the wells, in fact maybe a little more of the of the P.30.

1 colony had grown on the agar plate with both the vector and insert. Using a pipette tip the colony was scrapped and transferred into into 5ml of LB with 100 microG of ampicillin. This was then incubated over night at 37 degrees Celsius shaking.

3/12/2009 -

The Plasmid DNA was purified using the QIAprep spin mini-prep kit as per the provided protocol.

The purified DNA was then digested to check the insert had been incorporated into the plasmid, as follows:

2 microL DNA
2 microL H3 enzyme
2 microL H3 buffer
14 microL water

Then incubated at 37 degrees Celsius for 1 hour.

An electrophoresis gel was then ran using 5 microL of a 1kb+ ladder and 10 microL of the digested DNA. This was left to run at 110V for 1 hour.

The gel was then stained with ethidium bromide and a gel picture was taken. The vector and insert could not be seen, this may be due to a low concentration of DNA. A nanodrop was used to measure the DNA concentration:

-15.4 ng/microL

This is a very low yield usually you would expect a yield of around 200ng/microL using the QIAgen prep kit.


4/12/2009 -

Cells were counted using a haemocytometer. After counting it was seen there had been 2.4 population doublings. 300,000 cells were passaged to P.32.

The other flask was used to harvest cells to extract RNA and protein. This was done using the same method as previously.

7/12/2009 -

The clone with the low DNA yield was re-transformed. Three colonies were picked and transferred into LB + ampicillin and stored in the shaking incubator over night at 37 degrees Celsius.

8/12/2009 -

The DNA concentration of each of the three colonies was tested using the Nanodrop: -
1) 106.6 ng/microL
2) 123.9 ng/microL
3) 140 ng/microL

These concentrations meant there is a better yield of DNA and so a digestion would be more efficient.

Digestion of the plasmid DNA was done as follows: -

4 microL DNA
2 microL H3 buffer
2 microL H3 enzyme
12 microL water

This digestion was then run on a gel for 1 hour.

6 colonies of the re-transformed plasmids were put in LB and ampicillin and incubated over night at 37 degrees, shaking.

9/12/2009 -

DNA concentration of the 6 colonies (in ng/microL): -

1) 10.1
2) 6.3
3) 17.9
4) 10.6
5) 9.0
6) 14.0

These concentrations are too low to carry out a digestion.

9/12/2009 -

I met with Tracey to discuss how best to set out my report. We discussed how to set each part of the results into sections and to try and outline the project as soon as possible so results can be fitted in as we do the experiments.

Over the christmas holidays I will write up the introduction and begin the results using those we have collected already. I will also set out the outline for the rest of the report.

Ligations, Plating, Western Blots and Taqman continued

27/11/09 -

A transformation for each ligation was set up as before, again using DHSalpha bacteria. Once plated the dishes were incubated at 37 degrees Celsius.

30/11/09 -

A western blot was run to test the quality of the Zmpste24 antibody. Two gels were used together, a lower gel and stack gel, to create a 10% acrylamide gel. The gel was made as follows:

Lower Gel:

- 3 ml acrylamide

- 2.5 ml resolving buffer (TRIS-SDS, pH 8.8)

- 4.5 ml water

- 50 microL APS

- 6.8 microL TEMED (a catalyst)

The lower gel was levelled using isopropenol which does not mix with the gel but will sit on top.

Once the gel has set pour off isopropenol.

Stacking Gel:

- 0.65 ml acrylamide

- 1.25 ml resolving buffer (pH6.8)

- 3 ml water

- 25 microL APS

- 5 microL TEMED

Pour over lower gel and leave to set.

The protein concentration of P.29 and P.30 was measured at approximately 2 microG/microL using a BIO-RAD protein assay which had been diluted 1 in 5 ( 200 microL BIO-RAD in 800 microL water).

25 microL of each protein (equal to 50 microG of protein) was loaded onto the gel along with 5 microL loading dye. A 10 microL sample from SW1353 cells was also loaded. The gel was run at 150V for 1 hour.

Once the gel had finished running the proteins were transferred to a PDVF membrane. This was done as follows:

- Wash membrane in methanol (be very careful to not touch membrane, even with gloved hands, use tweezers)

- Wash in transfer buffer to remove methanol

- Soak filters in transfer buffer

- Transfer one filter onto transfer blot

- Carefully place membrane on top of filter

- Place gel (with stack gel cut off) on top of membrane

- Place another soaked filter on top of the gel

- Place electrode over the top

- Run at 15v for 30 minutes

Once gel has run it needs to be stained with the primary and secondary antibodies. The primary antibody is specific for Zmpste24 and will stick to it on the membrane. The secondary antibody is specific for the primary antibody and will anneal to it. On the secondary antibody is a bioluminescent which will allow for the visualisation of the Zmpste24 protein on the membrane.

When the gel has finished running block in 6% milk (3g Marvel in PBS Tween, a buffer with detergent in it) for 1 hour.

Then the primary antibody is added. The membrane was left in the cold room at 4 degrees Celsius over night.

Once again no colonies had grown on the plates with the vector containing the insert. This could be due to my pipetting technique so we decided to run one more ligation and transformation with Tracey helping me to ensure the right concentrations are used. Only one ligation was done using 10microL of insert and 1 microL vector with the same concentrations of buffer and ligase as before. The final volume was brought to 20 microL using water. The ligation was incubated at 16 degrees Celsius over night in the PCR machine.

The vector only plate did have colonies this time. This shows the ligation is working.

1/12/09 -

The membrane with the primary antibody was washed in PBS Tween twice shaking each time for 5 minutes. The secondary antibody was then applied then washed with PBS Tween twice for 5 minutes, shaking.

Another transformation was done using the ligation made the previous day as well as the DNA made from the complimentary miR-34a positive control. The same method was used as before.

When the membrane had finished washing a chemiluminescent substrate (1ml mixed with 9 ml water to give a 1 in 10 dilution) was poured over the membrane and left for 1 minute.

The excess was drained off and the membrane put in a light proof cassette. Once in the dark room an Auto-RAD sheet was put in the light proof cassette and left to expose for 1 minute then developed.

We got a good exposure after 1 minute so decided to do another one for 10 minutes, which gave an even better picture.

The picture showed a clear band for Zmpste24 at 50kDa for both MRC5 P.29 and MRC5 P.30 cells where as no band could be seen for the SW1353 cells (although this may have been because we loaded a smaller volume for these cells). It appeared that P.30 cells had a lower concetration of Zmpste24 as the band was much lighter compared to that of the P.29. This may be because the protein concentration loaded was different but it may also be because the P.30 cells contain less Zmpste24.

To see whether the protein concentration loaded was the same we will strip the antibodies used for Zmpste24 and will re-stain for GAPDH, a protein found in all cells. If the bands are the same size when staining for GAPDH it means the same amount of protein was loaded.

The Zmpste24 antibodies were stripped using 0.2M NaOH which was poured over the membrane and left shaking for 5 minutes. The membrane was then washed with water then PBS Tween shaking for 5 minutes each time. Then the membrane was blocked in 6% milk for 1 hour. Once the membrane had finished blocking the GAPDH primary antibody was added and the membrane left in the cold room, shaking, over night.

Next, I worked with Caroline to carry out a Taqman experiment. Using Taqman will allow me to determine the level at which a number of cell lines are expressing Zmpste24 and will allow me to compare between healthy and cancer cell lines.

17 cell lines were used:

Caroline reverse transcribed the RNA in each of the samples to give cDNA. But the cDNA was too concentrated to use for Taqman so it needed to b diluted 1 in 20 which was done by using 10 microL cDNA in 190 microL water. The Taqman plate was loaded as follows:

2 microL of each sample was loaded into wells A1-C10 as shown above and then made up to 10 microL with water.
Wells E1-G10 were loaded with 10 microL of each sample as shown. Wells D3-8 were loaded with an 18S standard curve and wells H3-8 were loaded with a Zmpste24 standard curve. Then each well was made up to 25 microL with Mastermix, forward primer, reverse primer, water and either the 18S probe (wells A1-D8) or the Zmpste24 probe (wells E1-H8).

Once each well had been loaded the plate was put in the PCR machine and run for 1 and a half hours.

Ligations, Plating, Western Blots and Taqman

23/11/09 -

Agar plates were made up by melting agar which then had 100 microL ampicillin added. The agar + amp was poured into petri-dishes and left to set.

While the agar plates were setting a transformation for each ligation was set up as follows: -
- Add 10 microL ligation to 50 microL DHSalpha (competent bacteria which had been treated with calcium chloride)
- Leave on ice for 15 minutes
- Place in 40 degrees Celsius water bath for 1 minute
- Place on ice for 2 minutes
- Add 500 microL LB
- Incubate at 37 degrees Celsius for 45 minutes, shaking.
- Spin for 2 minutes at 13,000 rpm
- Remove most of the LB, leave about 100 microL
- Resuspend very gently
- Pipette onto agar plate and spread with sterile spreader
- Incubate over night at 37 degrees Celsius.

24/11/09 -

No colonies had grown on the vector only agar plates and only a few grew on the other plates.

These few colonies were picked and added with LB with 100 microL ampicillin added.
These were incubated over night at 37 degrees Celsius, shaking.

25/11/09 -

Another digest was carried out as before. The digested vectors were then phenol chloroform cleaned as before. The vector was then run on an electrophoresis gel to check it was present.

The band was cut out of the gel and purified using the same method as previously.

26/11/09 -

Worked in cell culture today with Alba Warn to passage my cells at a particular density. It was decided plating 300,000 cells per well was sufficient. By plating at a known density it will allow me to determine how many population doublings have occurred between each passage.

The cells were counted using a Hemocytometer.

Results:

65 cells over 5 sections of the hemocytometer
65/5 = 13
13 x 10,000 = 130,000
So, 130,000 cells per ml
Cells were resuspend in 5ml so, 650,000 cells in total.

Need to plate 300,000 cells so need to take 2.3ml and transfer to new flask. This allowed for two new flasks to be passaged into P.31. The final volume was made up to 10ml using medium.

The remaining P.30 cells were passaged to P.31 and will be left to become confluent. When the cells are confluent they will be harvested and frozen down in case the cells become infected again.

The other flask of P.30 cells was used to harvest protein and RNA as follows:

- Aspirate medium
- Wash with PBS
- Trypsinise
- Incubate for a few minutes
- Resuspend cells in 1ml PBS
- Transfer 500 microL into two eppendorfs (one for RNA extraction, one for protein extraction)
- Spin at 13,000 rpm for 5 minutes

The protein extraction was carried out as before. For the RNA extraction the pellet was resuspended in 500 microL of TRIzol and frozen for the extraction to be completed another day.

Another ligation was set up as follows: -

Ligation	Insert	Vector	Ligase	Buffer	Water
	μL
1	2	1	2	2	13
2	5	1	2	2	10
3	10	1	2	2	5
4	0	1	2	2	15

The complimentary sequence for miR-34a also arrived this week so we are now able to set up a positive control for the ligations

Firstly they need to be made into DNA, which will be treated the same way as a PCR product. This was done as follows:

- Add 10 microL of forward oligo to 10 microL of reverse oligo and 30 microL water
- Boil on hot block for 5 minutes
- Leave to cool naturally (this allows for strands to anneal and create DNA)