• bring up about 1.5 µg total RNA in 17 µl volume in RNase free water;
• add 2 µl 10x DNaseI buffer;
• add 1µl DNase I (2 U/µl);
• mix gently and incubate for 15 min. at room temperature;
• add 2 µl 25mM EDTA and heat for 10 min @ 65°C (heatblock or waterbath);
• place on ice for 1 min;
• spin in centrifuge to collect reaction mix at the bottom of the tube;
• remove an aliquot for cDNA synthesis and store the remaining RNA in the -20° or -80°C freezer.

2. cDNA synthesis

For each reaction set up the following mix in 0.5 ml RNase-free microcentrifuge tubes and a total volume of 20 µl.

• 1X MM in 0.5 ml RNase free tube mix (total reaction volume is 20µl)
  • 4 µl 5x RT buffer
  • 12 µl treated RNA
  • 1 µl oligo dT 20 µM (final conc. 1 µM)
  • 1 µl dNTPmix 10 mM (2.5 mM each)
  • 1 µl Super RNAse.OUT (40 U/µl)
  • 1 µl of SuperScript II RT
• mix gently and collect by brief centrifugation if necessary
• incubate @ 42°C for 45-60 min
• optional: terminate reaction at 70°C for 5 - 15 min
• store cDNA at -70°C for several months or at -20°C for up to 1 week

3. Polymerase Chain Reaction (PCR)

Before you start. make sure the Thermal Cycler is programmed and the block temperature is at 80°C. Set-up five tubes. Tube # 1 should contain untreated RNA, tube #2 treated RNA and tube #3 cDNA, tube #4 genomic DNA and tube #5 water (negative control).

Each reaction (final volume of 50 µl) should contain the following:

• 35.5 µl ddH2O (up to 50 µl total volume)
• 5 µl 10x PCR buffer
• 5 µl 25 mM MgCl2
• 1 µl dNTP mix (2.5mM each)
• 0.5 µl primer 1 (10 µM)
• 0.5 µl primer 2 (10 µM)
• 0.5 µl Taq Polymerase (5U/µl)
• 2.0 µl cDNA/RNA/genomic DNA

Set-up a master-mix (MM) for five reactions (5xMM). That means pipette 5x the volume of individual solutions together in one centrifuge tube but omit the cDNA/RNA/genomic DNA. Mix the contents gently by flicking the tube and quick-spin to collect the solution. Next, transfer 49µl in to clean microcentrifuge tubes. Add 2 µl DNA/RNA, mix and top with 100 µl mineral oil.

• mix MM gently and quick spin to collect reaction mix at the bottom of the tube;
• transfer an 48 µl aliquot into clean 0.5 ml microcentrifuge tubes;
• add 2 µl cDNA/RNA/ genomic DNA, mix by flicking and quickspin; don't add anything to the negative control tube;
• top reaction mix with mineral oil;
• place tubes into PCR machine (80°C)
• set cycling: 1x (94°C for 2 min), 30x (1 min @ 94°C, 1 min @ (desired annealing temperature - usually 2°C below the primer annealing tempo.) °C, 1 min @ 72°C), 1x (72°C for 7 min), followed by cooling to 4°C;
• start thermal cycler; end of day 1.
• agarose gel electrophoresis of PCR reactions