NOTE: The following protocols and reagents are from Acturus. The final products can be used for RT-PCR or Microarray analysis.

Protocol 1:  Laser Capture Microdissection Using Frozen Tissue Sections

1.       Prepare your cryostat sections to a thickness of 7 mm. The sections can be used immediately or stored at –80 degrees for late use.

2.       Air dry the sections for 1 min and then fix sections in 70% ethanol for 1 min.

3.       Dip the sections in DEPC-treated H₂O for 1 min to remove OCT.

4.       Stain the sections with Hematoxylin solution for 1 min.

5.       Briefly dip the sections in DEPC-treated H₂O.

6.       Dehydrate the sections in 75%, 95% and 100% Ethanol for 1 min each.

7.       Clear the sections in xylene for 5 min.

8.       Dry the sections in a hood for 5 min.

9.       Capture interested cells using Laser Capture Microscope.

Protocol 2:  Total RNA Isolation from LCM-captured cells

1.       Pipette 50 ml Extraction Buffer in 0.5 ml microcentrifuge tube 

2.       Insert LCM cap into the tube

3.       Invert the tube and tape the tube to make sure the buffer covers the cap (you can change several caps in one tube)

4.       Incubate assembly for 30 minutes at 42 degrees

5.       Centrifuge assembly at 800xg for 2 minutes.

6.       It is okay to stop here, save tube at –80 degrees or Proceed further

7.       Pipette 250ml Condition buffer to column and wait for 5 minutes

8.       Centrifuge at 16000 xg for 1 min

9.       Pipette 50 of 70% ethanol into the cell extract and mix

10.   Pipette the cell extract mixture into the prepared column

11.   Centrifuge 2 min at 100x g and followed by 30 seconds at 16000g

12.   Pipette 100 ml wash buffer 1 and centrifuge 1 min at 8000 x g

13.   Pipette 100ml wash buffer 2 and centrifuge 1 min at 8000 x g

14.   Recentrifuge at 16000 g for one minute

15.   Transfer the purification column to a new microcentrifuge tube

16.   Pipette Elution Buffer directly onto the membrane (elution buffer volume can be 11 or 30ml) and incubate at room temperature for 1 min centrifuge the column for one minute at 1000g and followed by 16000 x g for 1 min      

Protocol 3:  RNA Amplification Using T7-based in Vitro Transcription

1. Total RNA –up to 5 mg
2. Add T7 oligo primer –1 ml
3. Add nuclease-free water to a final volume of 12 ml
4. Incubate at 70 degrees for 10 min
5. Add the followings:

10X first strand buffer        2  ml
Ribonuclease inhibitor      1  ml
dNTP                              4  ml
 

6. Transfer this mix to the RNA tube and mix well and put it in 42 degrees
7. Working quickly, add 1 ml reverse transcriptase to each tube and mix and replace the tubes immediately to reduce heat loss
8. Incubate for 2 hours
9. Add following Master Mix:

             Nuclease-free water             63 ml
            10X second strand buffer       10 ml
            dNTP Mix                             4  ml
            DNA polymerase                   2  ml
            RNase H                              1  ml
 

10. Incubate for 2 hours at 16 degrees
11. cDNA purification
12. Preheat Nuclease-free water to 50 degrees at least 10 min
13. Equilibrate cDNA filter 50 ml  cDNA binding buffer and incubate at RT for 5 min (do not spin)
14. Add 250 ml  of cDNA binding buffer to each cDNA sample and mix well
15. Apply mixture to an equilibrated cDNA filter cartridge
16. Spin at 10,000xg for 1 min
17. Discard the flow-through and replace the cDNA filter cartridge in 2 ml wash tube
18. Wash the cDNA filter cartridge with 650 ml cDNA wash buffer
19.  Spin at 10,000 for 1 min
20. discard the flow-through and spin the cDNA filter cartridge for additional 1 min
21. Elute with 10 ml of preheated water (50 degrees)
22. Leave at RT for 2 min and then centrifuge for 1.5 min at 10,000g
23. Elute with second 10 ml water (total about 16  ml)
24. Checking the volume to see if it is about 16-18 ml or less, add water to bring it to 16 ml If desired, the experiment can be stored at –20 degrees at this point
25. In vitro transcription to synthesize aRNA
26. To prepare the following mix (total 40ml)

                ds-cDNA                  16  ml
                ATP                           4 ml
                CTP                           4 ml
                GTP                           4 ml
                UTP                           4 ml
                T7 10x reaction buffer  4 ml
                T7 enzyme mix           4 ml
        

27. Incubate the reaction for 6-14 hours at 37 degrees using hybridization oven.
28. Add 2 ml Dnase I to each reaction and mix and incubate for 30 min at 37 degrees
29. Add 60 ml elution solution to each aRNA sample to bring the final volume to 100 ml and mix thoroughly
30. aRNA purification
31. Preheat water to 50 degrees for at least 10 min
32. Add 100 ml aRNA binding buffer to equilibrate aRNA filter cartridges for each sample  for 5 min at RT and do not spin
33. Add 350 ml of aRNA binding buffer to each aRNA binding buffer to each aRNA sample and mix thouroughly
34. Add 250 ml of ACS grade 100% ethanol to each aRNA sample and mix thoroughly
35. Pipet each sample mixture from above onto the center of equilibrated aRNA filter cartridge
36. Cenrtifuge for 1 min at 10.000 g
37. Wash the aRNA filter cartridge with 650 ml RNA wash buffer
38. Spin at 10.000 g for 1 min
39. Spin for additional 1 min to get rid of ethanol
40. Elute in elution solution if you intend to do a single round of amplification or for long tern storage (for several weeks); elute in Nuclease-free water if you intend to proceed to a second round of amplification, or you want to concentrate the aRNA by vacuum centrifugation
41. Add 50 ml  eluting solution or water to the center of the filter and leave at RT for 2 min
42. Spin at 10.000 g for 1.5 min
43. Repeat the elution with a second 50 ml prheat water or solution

(*Second Round Amplification)

44. Mix the aRNA sample with second round primers and bring the volume to 12 ml
45. Incubate at 70 degrees for 10 min
46. Put tubes on ice briefly
47. Prepare the following mix

                10X first strand buffer              2  ml
                Ribonuclease inhibitor            1  ml
                dNTP                                    4  ml
                Reverse transcriptase             1  ml
 

48. Incubate at 42 degrees for 2 hours
49. Add 1 ml RNase H  and incubate for 30 min at 37 degrees to remove aRNA
50. Add 5 ml T7  Oligo dT primer to the first strand reaction tube from step above
51. Incubate at 70 degrees for 10 min
52. Place the sample on ice briefly
53. Add the second strand cDNA synthesis reagents in the order listed

            First strand cDNA +T7 primer         26 ml
            Nuclease-free water                       58 ml
            10x Second Strand Buffer              10 ml
            5 mM dNTP mix                             4 ml
            DNA polymerase                            2 ml
           

            Total                                           100 ml
 

  54. Incubate for 2 hours at 16 degrees

55. Continue with labeling procedures