Protocols
1) Isolation of RNA
We use Trizol (Invitrogen) according to the manufacturer's instructions.
2) Quantification of RNA by 260/280 nm spectrophotometry
3) cDNA synthesis from 0,5 µg of total RNA
In our hands, Iscript (Biorad), Superscript (Invitrogen) both work well.
4) Run real-time PCR reactions; include standard curve
As a standard, we run 10 mL reactions, which saves reagent and does not reduce the quality. The protocol is here.
5) How to make a standard
We PCR the fragment which we intend to amplify and clone it into a standard vector using a PCR cloning kit (for instance Fermentas). For the product, we aim for a amplification size of 150-250 bp. Larger sizes will affect the efficiency of the reaction. We use primer Primer3 for primer design. Following isolation of a single clone, we verify the sequence and prepare a stock of the plasmid using a kit based on column ion-exchange (Promega Wizard, Fermentas, etc; the alkaline lysis method is less suitable, because the remaining RNA makes quantification by spectrophotometry difficult).
For the standard curve we linearise the plasmid and use 1 mg for making a 1:000 dilution. It is further diluted 1:10 to make a total of 8 standards ranging from 1 ng/mL - 1 fg/mL .
6) Normalise results relative to housekeeping gene
Since you usually want to compare samples, you need to be able to relate your result to parameter which allows you to normalise your results so they are independent on the number of cells which were isolated, the RNA recovery, the cDNA synthesis efficiency or other factors which affected your results that particular day. This is usually done by quantification of a housekeeping gene as described.
7) Delta Ct or standard curve?
If you choose to use the delta Ct method, you need to establish the efficiencies of your target cDNA amplification as well as of the housekeeping gene you use for standardisation. That takes a little effort, but can be done from the slope of a dilution curve.
We find it easier to run a standard curve with all amplifications. From the standard curve the number of copies in all samples are determined. If you use this method, you do not need to take the effeiciency into account.
8) Should you DNAse treat your samples?
We don't. We have no amplification, if we do not add reverse transcriptase to the cDNA reaction, so we have no DNA contamination.
