Author: SC, Patti Lab
This is performed as a 2-step reaction:
1. cDNA synthesis from DNase 1-treated total RNA
2. PCR
1. cDNA synthesis (Advantageä RT-for-PCR Kit - Clontech)
All reagents listed are provided with the kit
a) Quickly thaw each tube in kit and place on ice
b) Spin each tube briefly in a tabletop microcentrifuge and return to ice
c) In a sterile 0.5ml microcentrifuge tube, add purified DNase 1 -treated RNA preparation to a volume of DEPC-treated H2O that will give a total volume of 12.5 ul. (Can use 0.2-1ug of total RNA, but recommend 1ug where possible). Use the same amount of RNA for each sample.
d) Add 1ul of either the random hexamer or the oligo(dT)18 primer (both are
provided with the kit). (Oligo(dT) priming is the method of choice; however, if the 5¢ gene-specific Taqman primer is located greater than about 2-3 kb from the poly-A tail would recommend use of random hexamer)
e) Heat total RNA at 70°C for 2 min and RAPIDLY place heated RNA in ice
f) Add the components listed in the table belo w according to the volumes given
N.B. If more than one RNA sample is being used for RT-PCR, it is recommended to prepare a master reagent mix.
Some reactions may also be included substituting DEPC-treated H20 for reverse transcriptase, and later amplified in a separate PCR reaction in order to detect any genomic DNA contamination that may be present within the RNA samples.
g) Mix the contents of the tube by pipetting up and down
h) Incubate the reaction at 42°C for 1 hour
i) Heat at 94°C for 5 min to stop the cDNA synthesis reaction and to destroy any DNase activity; then spin down the contents of the tube
j) Dilute the reaction to a final volume of 100ul by adding 80ul of DEPCtreated H20. Vortex and spin again.
2. PCR protocol (multiplex reaction)
Before the PCR can be performed on the newly-synthesised cDNA in 1., a number of steps must be carried out:
a) Selection of a suitable internal standard
b) Design of primers and probes to target genes
c) Validation of equal efficiency of amplification of target gene and internal standard
d) Optimisation of primers
a) Internal standard selection
An appropriate gene for use as an internal standard and normalisation of data must be selected (this may be already designed and available from Applied Biosystems or can design own using PrimerExpress software)
N.B. For accurate quantitation, the internal standard must:
· be amplified simultaneously with the target
· be expressed at a constant level and be unaffected by the experimental treatment
· should be expressed at roughly the same level (preferably slightly more abundant – see primer optimisation below) as the RNA under investigation, to avoid competition of the more abundant target for PCR reagents
