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.