RNase Protection Assay
For quantitating RNA levels from either polyA+ selected RNA or crude RNA preps.
Solutions
10X Hybridization Buffer0.4 M PIPES 12.1 g PIPES (Sigma #P6757)
4.0 M NaCl 23.4 g NaCl
10 mM EDTA 2.0 ml 500 mM EDTA
pH to 6.7 and bring up to 100 ml with DEPC treated Q, autoclave1X Hybridization Mixture
8 ml formamide
1 ml DEPC treated Q
1 ml 10X Hybridization Buffer2X RNase Buffer
20 mM Tris 7.5 2 ml 1M Tris 7.5
10 mM EDTA 2 ml 500 mM EDTA 8.0
0.6 M NaCl 12 ml 5 M NaCl
up to 100 ml DEPC treated Q,
autoclave20% SDS
20 g SDS
up to 100 ml with QDEPC Treated Q
1 liter mili Q water
1 ml DEPC
shake overnight at 37°
autoclave twiceAlso Needed:
Proteinase K (10mg/ml in Q), RNaseA (2mg/ml), RNaseT1 (100 mg/ml).
Procedure
• To make the riboprobe linearize the appropriate template (5 mg) with the appropriate enzyme (5' overhang is best). Phenol/chloroform extract, EtOH ppt, wash and dry. Resuspend in 10 ml DEPC treated Q.
• Mix the following:T7 SP6 ml DEPC Q 3 ml DEPC Qml 10X Pol. Buffer (NEB) 4 ml 5X Pol. Buffer (Promega)ml 10 mM ATP 1 ml 10 mM ATPml 10 mM GTP 1 ml 10 mM GTPml 10 mM CTP 1 ml 10 mM CTPml 0.05 mM UTP 1 ml 0.05 mM UTPml RNaseIN 1 ml RNaseINml Template (0.5 mg) 1 ml Template (0.5 mg)ml a32P UTP (800 Ci/mmol) 5 ml a32P UTP (800 Ci/mmol)ml T7 RNA Polymerase (NEB) 1 ml SP6 RNA Polymerase (Promega)ml 100 mM DTT
Incubate @ 37°C 1 hour Incubate @ 40° 1 hour
• Add 1 ml DPRF DNaseI and incubate at 37°C for 15 minutes, repeat.
• Phenol/chloroform extract and EtOH precipitate with tRNA. Wash and dry. Resuspend in 1X Hybridization Mix.
• Dry down 5-20 mg RNA (crude) and resuspend in 29 ml 1X Hybridization mix. Add 1 ml riboprobe and incubate at 48°C overnight.
• Make the RNase Mixture and add 380 ml per tube. Incubate for 30 minutes at room temperature. ml 84 ml 126 ml 168 ml RNaseAml 84 ml 126 ml 168 ml RNaseT1
• Add 10 ml 20% SDS, 10 ml Proteinase K and incubate at 37°C for 15 minutes. Phenol/chloroform extract, and remove 360 ml of the aqueous phase. Add 1 ml EtOH (no salt required) and 1 ml tRNA. Spin, wash and dry.
• Resuspend in 10 ml Formamide Loading Dye and run on a 7-8% sequence gel (see protocol O.1).
