9. Protocols
- 9.1 Isolation of RNA
- 9.2 Quantification of RNA by 260/280 nm spectrophotometry
- 9.3 cDNA synthesis from 0,5 µg of total RNA
- 9.4 Run real-time PCR reactions; include standard curve
- 9.5 How to make a standard
- 9.6 Normalise results relative to housekeeping gene
- 9.7 Delta Ct or standard curve?
- 9.8 Should you DNAse treat your samples?
9.1 Isolation cialis over the counter< of RNA
We use Trizol (Invitrogen) according to the manufacturer’s instructions. It does not smell good, but it is by far the chepest RNA isolation kit available. You can even make it up yourself (write us for protocol), and it works for small amounts of tissue as well.
9.2 Quantification of RNA by 260/280 nm spectrophotometry
We use the Nanodrop spectrophotometer. It gives you, in addition to the 260 and 280 nm absorbance, a continues reading of wavelengths which allows you to distinguish “good” from “bad” RNA. At some occasions we have tested the Bioanalyzer for this purpose. We have not conducted a systematic comparizon, but experiensed that the readings from this instrument did not correlate well to denaturing agarose gel visualizations of the same samples. The Bioanalyser is useful for evaluation of the RNA quality as it visualizes the capillary electrophoresis result as an “electropherogram” which looks like a gel picture. However, we find no need of performing a more thorough analysis of the RNA quality than provided by the Nano-drop, which is therefore the only RNA analysis that we perform before we use the RNA for cDNA synthesis.
9.3 cDNA synthesis from 0,5 µg of total RNA
In our hands, i-Script (Biorad), qScript (Quanta) and Superscript (Invitrogen) all work well, but again – check the price per reaction. They are quite variable. We usually scale the reaction down to 10 µl.
9.4 Run real-time PCR reactions; include standard curve
As a standard, we run 10 µl reactions, which save reagent and do not reduce the quality. The protocol is here.
9.5 How to make a standard
We PCR the fragment in question. For the product, we aim for a amplification size of 150-250 bp. Larger sizes will affect the efficiency of the reaction. We use Primer3 for primer design. We verify the sequence. You may also clone it into a standard vector using a PCR cloning kit (for instance Fermentas) and prepare a stock of the plasmid (preferentially by affinity-matrix purification: Promega Wizard, Fermentas, etc; the alkaline lysis method is less suitable, because the remaining RNA makes quantification by spectrophotometry difficult, but if you use it, quantify on gel). If using plasmid, we linearise the it and use 1 mg to make a 1 in 1:000 dilution. It is further serially diluted 1:10 to make a total of 8 standards ranging from 10E-3 to 10E-11 diutions. The dilutions are the same for the PCR products.
9.6 Should I normalise my results relative to a housekeeping gene?
Yes. Since you usually want to compare samples, you need to relate your result to a parameter which allows you to normalise your results so that 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.
9.7 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 online viagra in canada< 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 efficiency into account.
9.8 Should you DNAse treat your samples?
We don’t. We have no amplification, if we don’t add reverse transcriptase to the cDNA reaction, so we have no amplifiable DNA contamination.