Yeast RNA extraction & qRT-PCR

  1. Prepare 2% agarose gel for RNA.
  2. For 40mL RNA gel solution, add DEPC water 17.64mL in a small beaker.
  3. Add agarose 0.48g in the tube.
  4. Melt agarose completely by microwave.
  5. Be careful because it can boil. First heat for 30s, then heat for 10s several times until agarose is completely melted.
  6. Add 4mL of 10X MOPS and stir it.
  7. After cooling the gel solution down to around 50C, add 0.72mL of 37% formaldehyde and stir.
  8. Pour the solution to the gel caster.
  9. Prepare RNA running buffer as shown in the table.
  10. Perform DNase I treatment using the table below.
  11. Perform cDNA synthesis using the table below.
  12. Mix in PCR tube and incubate at RT for 10min.
  13. Incubate in PCR machine at 42C for 1hr and 85C for 5min.
  14. Perform qRT-PCR using the table below.

Tables / recipes

Reagent 20 mL
DEPC water 17.64 mL
Agarose 0.24 g
10x MOPS 2 mL
37% formaldehyde 0.36 mL
Total 20 mL
Reagent 20 mL 40 mL 80 mL
DEPC water 17.64 mL 35.28 mL 70.56 mL
Agarose 0.24 g 0.48 g 0.96 g
10x MOPS 2 mL 4 mL 8 mL
37% formaldehyde 0.36 mL 0.72 mL 1.44 mL
Total 20 mL 40 mL 80 mL
Reagent 100 mL 50 mL
10x MOPS 100 mL 50 mL
37% formaldehyde 20 mL 10 mL
RNase-free water 880 mL 440 mL
RNA 2 ug
10x buffer 5 uL
DNase I 2 uL
RNase inhibitor (RNase out) 1 uL
Total 50 uL