In vitro Transcription for RNA in situ Probes

(version 1.0;  01/27/04)

 

Purpose: To generate a labled RNA molecule complementary to a gen of interest for use in in situ hybridization.  Using this method we can visualize the expression pattern of the gene in an organism.

 

I.              Linearize plasmid. 

a.     Choose an enzyme that will cut the plasmid on the side opposite of the promoter being used to transcribe RNA.

b.    Set up restriction digest reaction (25 µl reaction)

12.5 µl purified DNA (or more up to 5 ug total)

2.5 µl 10X buffer

0.25 µl 100mg/ml BSA (optional)

1 µl enzyme

add sterile MilliQ H2O up to 25 µl

c.     Incubate at appropriate temperature for 2 hours to overnight.

d.    Stop reaction by incubating at 70 degrees for 20 minutes.

e.    Check digestion by running 1 µl on 0.8% agarose, 1X TAE gel.

 

II.            Transcribe RNA

a.     Choose the appropriate RNA polymerase.

                                                    i.     You will need to determine the orientation of your sequence in the plasmid.

                                                     ii.     RNA polymerases transcribe the compliment of the sequence.  If you are making an anti-sense probe you will want to choose the polymerase whose promoter is 5¹ of your sequence (when it is oriented so you could read the ORF).  Use the polymerase on the 3¹ side if you want to make a sense control probe.  Make sure you cut with an enzyme on the opposite side of the plasmid insert as your desired RNA polymerase promoter.

b.    Set up the in vitro  transcription reaction

                                    5 µl digested DNA

                                    4 µl 5X optimized transcription buffer (Promega #P1181)

                                    2 µl 100mM DTT (Promega #P1171)

2 µl DIG RNA labeling mix (Roche #1277073)

                                    X µl RNA polymerase (add total of 40 units)

            sterile MilliQ H2O to 20 µl

c.     Incubate at 37 degrees for 2 hours.

d.    Optional: Remove plasmid (DNA) template by DNAse digestion

                                                    i.     add 2 µl DNAse I, RNase-free (Roche #0776785)

                                                     ii.     incubate at 37 degrees for 30 minutes


e.    Precipitate RNA by adding

5 µl 0.1M EDTA pH8.0

            1 µl 1mg/ml Glycogen (Ambion #9510)

            3.1 µl 4M LiCl

            90 µl EtOH

 

f.      Mix and store at ­80 degrees for 90 minutes to overnight

g.    Spin at 14,000 rpm at 4 degrees for 15 minutes.

h.     Carefully remove supernatant and wash with 200 µl 80% EtOH.

i.       Spin 2 minutes at 14k rpm at RT.

j.       Remove supernatant.  Spin for 30 seconds and remove all traces of EtOH.

k.     Air dry 4 minutes.

l.       Resuspend in 100 µl sterile MQ H2O.  Vortex briefly every 2 minutes for 10 minutes to resuspend RNA.

m.   Run 4 µl synthesized probe on 0.8% agarose, 1X TAE gel along with 2 µl of DIG-labeled control RNA (Roche # 1585746) to estimate yield.