For general safety in laboratory work, consider the general document RWS 004 on environmental safety and work security.
2. Rationale
To quantitatively determine the mRNA expression of specific genes in cell/tissue samples
3. Application
To be performed by laboratory personnel or persons trained in real-time PCR after qualification by an instructor
4. Applicable documents
· Primer Express PRBI 002
· GeneAmp 5700 Sequence Detection System PRBI 001
5. Definition
PCR Polymerase Chain Reaction
6. Material
· GeneAmp 5700 Sequence Detection System (Applied Biosystems)
· MicroAmp Optical Tubes and Optical Caps, or
ABI PRISM Optical 96-well Reaction Plates and ABI PRISM Optical Adhesive Covers (Applied Biosystems)
· TaqMan Universal Master Mix (Applied Biosystems, cat. # 4324018): 2x concentrate, containing AmpliTaq Gold DNA polymerase, dNTPs with dUTP, passive reference, and optimized buffer components.
If contamination with carryover PCR products is suspected, use Universal Master Mix containing AmpErase UNG (Uracil-N-glycosilase), which hydrolyzes dU-containing DNA (Applied Biosystems, cat. # 4304437).
· TaqMan Probe (Microsynth): Oligonucleotide labeled with the reporter dye molecule FAM (6-carboxyfluorescein) at the 5´end and with the quencher dye TAMRA (6-carboxy-N, N, N´, N´-tetramethylrhodamine) at the 3´end. Dissolve to 5 µM with Tris-EDTA buffer (prepared from 100x concentrate (SIGMA cat. # T-9285) with DEPC-treated water) by incubation at 65℃ for 5 min, shaking, 65℃ for 3 min.
· Forward and reverse primers (Microsynth): Dissolve to 45 µM with Tris-EDTA buffer (prepared from 100x concentrate (SIGMA cat. # T-9285) with DEPC-treated water) by incubation at 65℃ for 5 min, shaking, 65℃ for 3 min.
For 18S ribosomal RNA: 18S rRNA primer/probe mix 20x (Applied Biosystems cat. # 4310893E)
· DEPC-treated water (see PRBM 006)
7. Procedure
Protocol for 25 µl reaction volume:
TaqMan Universal Master Mix 2x 12.5 µl → 1x final concentration
Forward Primer 45 µM 0.5 µl → 900 nM
Reverse Primer 45 µM 0.5 µl → 900 nM
TaqMan Probe 5 µM 1.25 µl → 250 nM
cDNA + DEPC-treated H2O 10.25 µl
Protocol for 18S rRNA (endogenous control):
· Prepare reaction mixture for (2n + 2) reactions (n=sample number) by combining Universal Master Mix, primers / probes, and DEPC-treated H2O
· Distribute into Optical Tubes or Optical Plates, use inner wells first
· Add cDNA samples (2–5 µl / reaction) in duplicates, run 1 NTC (no template control) reaction without cDNA for each reaction mixture
· Cover tubes with Optical caps or plate with adhesive cover
· Centrifuge briefly to remove air bubbles from the bottom of the tubes
· Place Cover Compression Pad onto Optical Plate
· Run PCR at standard conditions (2 min @ 50℃ if AmpErase UNG is used; 10 min @ 95℃ to activate DNA polymerase; 40 cycles of 15 sec @ 95℃, 1 min @ 60℃) on GeneAmp 5700 Sequence Detection System (see PRBI 001)
8. Documentation, Archiving
After the run the data are automatically saved on the local drive. It is recommended to save it also on the network.
During each cycle of the PCR the 5´→3´exonuclease activity of AmpliTaq Gold DNA polymerase cleaves the TaqMan probe, thereby increasing the fluorescence of the reporter dye at the appropriate wavelength. The increase in fluorescence (△Rn) is monitored during PCR. Threshold cycle (CT value) is the PCR cycle number at which the amplification curve reaches the threshold DRn.
· Set the threshold DRn within the exponential phase of the logarithmic scale amplification curve.
· Perform relative quantification of mRNA targets according to the comparative CT method (User bulletin #2, ABI PRISM 7700 Sequence Detection System, Applied Biosystems) with 18S ribosomal RNA as endogenous control: Quantity is expressed relative to a calibrator (baseline) sample. The amount of mRNA relative to the calibrator is calculated as 2-△△CT; △△CT is the difference between CT(target) – CT(control) of sample and CT(target) – CT(control) of calibrator.
9. Special Requirements, Quality Assessment
Verify the amplification efficiency of each primer/probe system by amplifying the respective gene as well as the 18S ribosomal RNA endogenous control of serial 1:2 dilutions of a cDNA sample in TE buffer. The difference △CTbetween the target gene and the endogenous control of each dilution is plotted versus log quantity of the cDNA (which is 1, 0.5, 0.25, 0.125, and so on). The amplification efficiency is acceptable, if the slope of the resulting graph is between -0.01 and +0.01, i.e. △CT does not show a clear trend, over a series of at least 5 dilutions.
10. References
User bulletin #2, ABI PRISM 7700 Sequence Detection System, Applied Biosystems
TaqMan Universal PCR Master Mix, Protocol, Applied Biosystems, 1998
