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).