RT-PCR is a procedure that makes it possible to compare concentration of different mRNA in the cell culture. The first step is to select the mRNA which concentration we would like to compare and to design the relevant primers. Primer design can be done with the [http://biotools.umassmed.edu/bioapps/primer3_www.cgi primer design tool] from the university of Massachussets medical school. When you've received the primers, prepare them by diluting with a TE buffer to a high concentration, then prepare a working solution (generally 1μM).

the concentrations of the messenger RNAs in a cell. Beo

PCR is a reaction that makes it possible (and relatively easy) to amplify a certain region of DNA. The first step is the selection of that region (and the design of the relevant primers). Primer design can be done by hand, or by using our Primer Design Helper. Once done, order the primers (in our case, we ordered from them IDT). When you've received the primers, prepare them and make sure you've got your PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). Start preparing your master mix, the composition for one tube is: 1X Mastermix 20μl reaction, add in this order

mRNA extraction is a procedure that makes it possible to recover all the messenger RNA of a cell. Before proceeeding further, make sure you have your mRNA extraction kit (we've used the RNeasy Plus Mini Kit from Qiagen and the QIAshredder Kit from Qiagen), all the necessary reagents (β-mercaptoethanol, 70% ethanol and 100% ethanol) and RNAse inhibitor to avoir RNA degradation (RNaseZAP form Sigma-Aldrich). Once you've got all the necessary equipment, proceed according to the hnadbook of the kit.

With the RNeasy Plus Mini Kit from Qiagen the procedure takes about an hour.

Prepare the cell culture

1. Harvest 0.5*10^6 cells from the culture.

2. Centrifugate 5 min at 300g

3. Discard the aliquot, resuspend the pellet in the PBS solution

4. Centrifugate 5 minutes at 300g

5. repeat step 3.

6. repeat step 4.

7. Discard the aliquot, keep the pellet.

Please pay attention to remove all the liquid, otherwise it might inhibit cell lysis.

Prepare the lysis buffer

Step to be performed if β-mercaptoethanol haven't been added yet to the lysis buffer.

1.Pipet 594μL of lysis buffer (Buffer RLT Plus) into a 1.5 mL eppendorf

2.Add 6μL of β-mercaptoethanol

Step 2 must be done in a fume hood, since β-mercaptoethanolis toxic and nocif upon inhalation.

Prepare your work place

RNA is easily degraded by the RNAses present on the surfaces, surface of skin or just dust particles in the air. To avoid contamination with the RNAses, please clean your workplace and all the instruments with 70% ethanol, then spray them with RNAse inhibitor. Change gloves whenever you touch any common surface and spray them with RNAse inhibitor regularly. Close all the tubes, eppendorfs and tips boxes as soon as you've done with pipeting to avoid contamination from RNAses on the dust particules.

Lysis buffer and β-mercaptoethanol are both toxic to the environment and the RTL buffer cannot be bleached. Please, anticipate a storage for both of them and dispose them in according with the instructions in the handbook.

mRNA extraction

(From the Qiagen RNeasy Plus Mini Kit [http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasyplusminikit.aspx#Tabs=t2 mini-handbook])

1. Loosen the cell pellet by flicking the tube

2. Add 600μL of the prepared lysis buffer

3. Vortex or mix vigurously

4. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at maximum speed

5. Transfer the homogenized lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube. Centrifuge for 30 s at ≥8000 x g. Discard the column, and save the flowthrough

6. Add 600μL of 70% ethanol to the flowthrough, and mix well by pipetting. Do not centrifuge.

7. Transfer 700 μL of the sample to an RNeasy spin column placed in a 2 ml collection tube. Centrifuge for 15 s at >8000 x g. Discard the flow-through. Reuse the collection tube in next step.

8. Add 700 μl Buffer RW1 to the RNeasy spin column. Centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in the next step.

9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥8000 x g to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in in the next step.

10. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at .8000 x g to wash the spin column membrane

11. Place the RNeasy spin column in a new 2 ml collection tube, and discard the old collection tube with the flowthrough. Centrifuge at full speed for 1 min.

12. Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μl RNase-free water directly to the spin column membrane. Centrifuge for 1 min at ≥8000 x g to elute the RNA.

13. Recover the flow-through, place it in the RNeasy spin column atop the same collection tube. Centrifuge for 1 minute at >8000 x g to finish the elution.

14. Characterize the obtained mRNA solution by nanodropping and store it at -80 until use.