Note: For the reverse transcription reaction, the final volume does not have to be exact, however for sample comparison, the amount of starting material (added RNA) MUST be equal.
5.1.5 Combine all reagents except RNA and mix thoroughly and spin down.
5.1.6 Aliquot master mix into either thin wall tubes OR a 96-well reaction plate.
Note: The BioRad iCycler iQ PCR plate is designed to be broken into four separate 24-well sections which is useful in place of individual tubes.
5.1.7 Add RNA samples to individual tubes or wells and seal.
Note: A weak seal of the tube/well could lead to evaporation during the reverse transcription and loss of reaction.
5.1.8 Mix tubes/plate and then spin down to remove any air bubbles.
5.1.9 Thermal cycling parameters:
Note: The incubation step is necessary to maximize primer-RNA template binding.
Note: Store all cDNA samples at –15 to –25℃
5.2 Real-Time PCR (for either Applied Biosystems SYBR Green Master Mix Kit or Qiagen QuantiTect SYBR Green PCR Kit)
5.2.1 Keep all reagents on ice during set up.
Note: It is not necessary to clean up the cDNA reactions for use in the PCR reaction.
5.2.2 Although an RNAse free environment is not necessary for the PCR of cDNA, a clean workspace is a good idea.
5.2.3 Mix 2X Master Mix thoroughly before use.
Note: The PCR step can be done in either a 50 ml or a 20 ml reaction volume for the 96-well plates with similar results and in a 20 ml reaction volume for the 384-well plates.
EITHER:
5.2.4 Master mix (for a single 50 ml reaction):
5.2.5 Add 49.0 ml of mix to the necessary wells in the 96-well plate. OR:
5.2.4 Master mix (for a single 20 ml reaction):
Final Concentration
(in ml)
5.2.5 Add 19.0 ml of mix to the necessary wells in the 96-well or 384-wellplate.
5.2.6 Add 1.0 ml of cDNA to the appropriate wells in the plate.
Note: Additional cDNA template may be added to the reaction if desired.
5.2.7 Seal plate and mix. Spin down to remove any bubbles in the wells.
Note: Samples for a standard curve should be included in EVERY real-time run for quantitation.
Note: Negative controls for every primer set used should be included to verify the absence of primer dimer amplification and contamination.
5.3 Thermal Cycling Parameters
5.3.1 For Qiagen QuantiTect SYBR Green PCR Kit
Dissociation/Melting Curve (machine specific)
5.3.2 For Applied Biosystems SYBR Green PCR Master Mix:
