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.