Ribonuclease Protection Assay

Ribonuclease Protection Assay

contributed by James McCaughern-Carucci, Yale University

Most RNase Protection protocols require an overnight hyb with numerous subsequent clean-up steps. This method requires a maximum hyb of four hours, and the clean-up steps are the barest minimum, yet still produce nice images. It is strongly recommended to titer RNase concentrations with probes prior to running experiments...some require more RNase than others.

Part I: In Vitro Transcription

In a sterile 1.5ml microfuge tube, combine the following:

("ul"= microliter; all reagents obtained from )

4 μl 5x Transcription BufferQ

2 μl 0.1M DTT

4 μl 2.5mM NTP's (A, C, G)

0.8 μl RNasin RNase Inhibitor (25U/ul) (Promega)

2.4 μl of 100uM cold UTP (Note: Use 1mM UTP for loading control probes e.g. B-actin)

1 μl of 1 ug/μl linearized DNA template

5 μl of 10 uCi/μl P32 UTP (800Ci/mmol) (Dupont NEN - #NEG507X), or 1 μl for loading control probes

1ul RNA polymerase SP6,T7 or T3 (concentration varies by vendor)

Total Volume ~20μl.

Incubate 1 hour @ 37℃.

Add 2ul of DNase I (Promega) to each transcription, incubate 20 minutes @ 37℃.

Part II: Probe Purification

Purify probes using QIAGEN QIAquick Nucleotide Removal Kit or Boehringer Spin Columns (G50 Sephadex).

Check 1μl in scintillation counter, P32 channel. A good probe will be ~ 5x105 to 1x106 cpm.

Part III: Hybridization

Turn heatblock on to 95℃.

For samples in water or ethanol, dry down appropriate amount of RNA, and include a tube with 1ul of tRNA or Glycogen (Sigma), this is the negative control.

Each sample should have the following:

24μl Formamide

2μl 0.6M PIPES

2.4μl 5M NaCl

0.3μl 0.1M EDTA

2 x105 cpm main probe

5 x104 cpm loading control probe

DEPC water

Total Volume 30μl

Mix samples well. Heat @ 95℃ for 10 minutes. Incubate 4 hours @ 55℃.