RNase Protection Assay--RNA酶保护
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For quantitating RNA levels from either polyA+ selected RNA or crude RNA preps.
Solutions
10X Hybridization Buffer
0.4 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, autoclave
1X Hybridization Mixture
8 ml formamide
1 ml DEPC treated Q
1 ml 10X Hybridization Buffer
2X 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,
autoclave
20% SDS
20 g SDS
up to 100 ml with Q
DEPC Treated Q
1 liter mili Q water
1 ml DEPC
shake overnight at 37°
autoclave twice
Also Needed:
Proteinase K (10mg/ml in Q), RNaseA (2mg/ml), RNaseT1 (100 m g/ml).
Procedure
• To make the riboprobe linearize the appropriate template (5 m g) with the appropriate enzyme (5' overhang is best). Phenol/chloroform extract, EtOH ppt, wash and dry. Resuspend in 10 m l DEPC treated Q.
• Mix the following:
T7 SP6
6 m l DEPC Q 3 m l DEPC Q
2 m l 10X Pol. Buffer (NEB) 4 m l 5X Pol. Buffer (Promega)
1 m l 10 mM ATP 1 m l 10 mM ATP
1 m l 10 mM GTP 1 m l 10 mM GTP
1 m l 10 mM CTP 1 m l 10 mM CTP
1 m l 0.05 mM UTP 1 m l 0.05 mM UTP
1 m l RNaseIN 1 m l RNaseIN
1 m l Template (0.5 m g) 1 m l Template (0.5 m g)
5 m l a 32P UTP (800 Ci/mmol) 5 m l a 32P UTP (800 Ci/mmol)
1 m l T7 RNA Polymerase (NEB) 1 m l SP6 RNA Polymerase (Promega)
2 m l 100 mM DTT
Incubate @ 37℃ 1 hour Incubate @ 40℃ 1 hour
• Add 1 m l DPRF DNaseI and incubate at 37℃ for 15 minutes, repeat.
• Phenol/chloroform extract and EtOH precipitate with tRNA. Wash and dry. Resuspend in 1X Hybridization Mix.
• Dry down 5-20 m g RNA (crude) and resuspend in 29 m l 1X Hybridization mix. Add 1 m l riboprobe and incubate at 48℃ overnight.
• Make the RNase Mixture and add 380 m l per tube. Incubate for 30 minutes at room temperature.
5 tubes 10 tubes 15 tubes 20 tubes
1.05 ml 2.1 ml 3.15 ml 4.2 ml 2X RNase Buffer
0.96 ml 1.9 ml 2.9 ml 3.86 ml DEPC Q
42 m l 84 m l 126 m l 168 m l RNaseA
42 m l 84 m l 126 m l 168 m l RNaseT1
• Add 10 m l 20% SDS, 10 m l Proteinase K and incubate at 37℃ for 15 minutes. Phenol/chloroform extract, and remove 360 m l of the aqueous phase. Add 1 ml EtOH (no salt required) and 1 m l tRNA. Spin, wash and dry.
• Resuspend in 10 m l Formamide Loading Dye and run on a 7-8% sequence gel (see protocol O.1).