An Introduction to qRT-PCR Assays Using Sybr Green (Using SDS ...

Reaction conditions:Initial step:Taq activation95°C 10 minutes40 cycles of:DenaturationAnnealing/Extension95°C 15 seconds60°C 60 secondsConfirmation of Product SpecificityAs primers alone determine the specificity of the assay, it is essential to confirm thatthe correct product is amplifiedThis can be accomplished in three ways: by gel electrophoresis, sequencing toconfirm product identity and melting curve analysis.1. Gel electrophoresis – run 10-15 ul of sample on agarose gel2. Sequence – cut band from gel, and direct sequence3. Melting Curve – using 25 ul of PCR product on SDS 7700 (takesapproximately 20 mins). Ramp temperature from 60 to 95°C and recordfluorescence. When this is plotted in ‘Dissociation Curve’ software asrate of change, this will highlight the melting temperature of thedouble-stranded product, and will indicate the presence of any primerdimers.Use a temperature just below the product melting peak forrecording fluorescence during real-time PCR run.Do not use primers for qPCR if you doubt their specificity!3. Extract RNACollect samples for analysis and either snap freeze or store in RNApreservation medium such as RNAlater (Ambion). We have used the latter forsome time and found it to work well.Extract RNA according to desired protocol – we typically useTriReagent from Sigma-Genosys to isolate total RNA. Either total RNA ormRNA may be used. We have found that homogenising using a Fast-Prephomogeniser system combined with a half volume Tri-Reagent extractiongives good yields – routinely around 4-6 ug of total RNA from paired retina,and up to nearly 20ug from paired eyes. We usually resuspend pelleted RNAin around 20 ul of RNase-free water, either DEPC or RNASecure treated (thelater is simply added to water and activated by heating to 60°C).