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.