"
Contents |
Protocol: RT-PCR
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 primer design tool from the university of Massachussets medical school. Once done, order the primers (in our case, we ordered from them IDT). 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). Make sure you've got your RT-PCR kit (we used the Power SYBR-Green RNA-to-CT 1 step Kit from Applied Biosystems) and the DNAses mix (we used the TURBO DNA-free™ Kit from Life Technology). Even though the DNAse mix is not necessary, it decreases the noise from the genomic DNA. Provide yourself with the 96 plates for the PCR (we used MicroAmp® Optical 96-Well Reaction Plates from Applied Biosystems) and plate cover film (we used MicroAmp® Optical Adhesive Film from Applied Biosystems).
Primer design
The primers for the RT-PCR should have a melting temperature between 56°C and 60°C (optimal at 58°C). The optimal length for the primers is 20 nucleotides with CG content between 40 and 60%. The final product should be about 100 bp long for the optimal results.
DNAse Treatment
Add 0.1 volume of the DNAse buffer to the mRNA solution
Add 1 uL of DNAse to the mRNA solution
Leave for incubation for 30 minutes at 37°C.
Add 0.1 volume of DNAse Inactivation Reagent to the solution, mix well by flicking. Do not centrifuge.
Leave for 5 minutes.
Centrifuge at 10 000 g for 1.5 minutes and transfer RNA to a fresh tube. Pay attention not to touch the beds, since it might inhibit the PCR reaction.
Master Mix
The composition for one 10.0μL tube is:
| Reagent | Volume [μL] |
|---|---|
| SYBR Green RT-PCR Mix | 5.0 |
| Forward primer (2μM) | 1.0 |
| Reverse primer (2μM) | 1.0 |
| RT enzyme | 0.08 |
| Rnase- free H20 | complete to 8μL |
2μL of template will be added upon addition to the 96 well plate to complete to a final volume of 10μL.
Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips)
Note : It is a good idea to prepare for each sample a master mix without the RT Enzyme, so that we can measure the degree of contamination with the genomic DNA.
Protocol
Get the 96 well plate and drop 2μL of template directly to the wells, then complete with 8μL of Master Mix to 10μL of total solution. Complete all the wells in rows (marked by letter), without leaving any column dropped out (it is better for the analysis).
Cover the plate with the plate cover film and get rid of all the bubbles by successively centrifugating - shaking the plate and centrifugating again.
Place the plate in the RT-PCR machine. Add an incubation step: 30 minutes at 48 degrees and configure it to run for 40 PCR cycles.
