Real-time reverse transcription qPCR (SOP_SSO_060910e)

Total RNA is extracted from 10 ml (OD: 0.9) of exponentially growing Sulfolobussolfataricus cells. Extraction is performed using mirVana total RNA extraction kit(Ambion). The amount of RNA is quantified by NanoDrop (PEQLAB) and thequality check was performed using an Experion machine (Biorad). From eachsample ~500 ng of RNA are treated with 10 U of DNase QR1 (Promega) in atotal volume of 10 μl and incubated 30 min at 37°C. The reaction is stoppedadding 1μl of DNAse sTOP solution and incubating at 65°C for 10 min. DNAcontamination is checked before cDNA synthesis by PCR using S. solfataricusspecific primers. After DNAse digestion 1 μl of random hexamer primers (50mM)and 1 μl of dNTPs (10 mM) are added to the solution and incubated 5 min at65°C. The samples are then incubated on ice for 5 min. After incubation 10 μl ofcDNA master mix is added to the RNA-dNTPs-primer mix ( 2 μl 10x FS buffer, 4μl 25 mM MgCl2, 2 μl of DDT 10mM and 10 U of SuperScriptIII Retrotrascriptase(Invitrogen)). The mixture is incubated at 25°C for 10 min followed by anincubation at 50°C for 1 h. For termination, the reaction is heated for 5 min at80°C. The RNA-cDNA hybrids are then digested using 5 U of RNAseH andincubating the samples at 37°C for 1 h. The cDNA samples are then diluted 1:10with DEPC water and stored at -20°C until use.

Standard curves have been prepared using serial dilution of gene specific longPCR fragments of 3.5 to 5 kb. The standards were cleaned from gels andquantified by NanoDrop 1000 (PEQLAB) using the formula number = (ng *number/mole) / (bp * ng/g * g/ mole of bp). From this the number of copies pernanogram (ng) of standard is assigned. Starting with the concentration of 5 x 107molecules/μl, serial dilutions are performed until a final concentration of 5 x 101molecules/μl. This serial dilution is used to obtain the standard curve necessaryfor the RNA quantification and primers efficiency calculation.

The primer used in the qPCR reaction have been designed using primer3software ( to amplify a region of 200-320nt withan optimal melting temperature of 60°C.

The qPCR reactions are carried out in the Real Time RealPlex machine(Fermentas) using the following qPCR reaction mixture: 10 μl Biorad Supermix(SYBR green), 1 μl 10 mM Primer mix, 7 μl PCR grade water and 2 μl of cDNA (5ng RNA). A first denaturation step of 5 min at 94°C is followed of 40 cycles: 94°C20”, 58°C 20”, 72°C 40”, and the fluorescence is measured each step at 76°C for30”. The specificity of the amplification is evaluated from dissociation curves,obtained after heating the samples for 3 min at 95°C and then measuring thefluorescence for 15” starting from the temperature of 45°C to 95°C with 1°Cinterval.

Each cDNA sample is analyzed in triplicate, and the fluorescence data areacquired using the RealPlex software (Fermentas). The number of copies ofeach gene is then calculated plotting the Threshold Cycle (Ct) of each cDNA onthe standard curve obtained from the Ct of serial dilution of the standard.The copy number of each gene per nano-gram (ng) of total RNA is calculateddividing the copy number obtained for the ng of total RNA added in each well.

Go back