TITLE: Optimized protocol for the RNA isolation of laser microdissected formalin-fixed paraffin-embedded uterine scar tissues for RNA expression analyses
AUTHORS: Alexander   Paping, Clara Basler  , Rebecca  C. Rancourt, Loreen Ehrlich, Kerstin Melchior, Wolfgang Henrich, Thorsten Braun

[DESCRIPTION]
Samples for histological analyses are often formalin-fixed paraffin-embedded (FFPE) and slide-mounted. This complicates RNA extraction for molecular downstream applications. Additionally, when the region of interest is limited to a smaller area with laser microdissection (LMD), extracting adequate quality and quantity RNA is difficult. This is a protocol for maximized RNA output from FFPE tissue devised to identify and analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars resulting from prior cesarean deliveries. Gomori Trichrome staining allowed for region identification for LMD. Successful RNA isolation, reverse transcription, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. This description provides an optimized step-by-step protocol that yields sufficient RNA for qRT-PCR analyses from challenging tissue such as LMD-FFPE samples.

[STEPS]
SECTION: Step 2: Tissue Processing
5. Treat surfaces and eqipment with RNase Away to prevent RNA degradation by RNases during tissue preparation.
SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
10. Before cutting, cool tissue blocks at 4°C for 1 hour to harden the wax and thus allow easier cutting.
SECTION: Step 7: Laser Microdissection and Scratch off
22. Put slide into the slide holder with the specimen facing downwards.
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
53. Prepare Ladder

SECTION: Step 11: qPCR (TaqMan techc)
67. 
Carry out qPCR  in Singleplex using Taqman probe technology in 96 well plates in an ABI 7500 (Applied Biosystems)  



SECTION: Step 12: Gel electrophoresis
70. Prepare TAE Buffer (1X):
SECTION: Step 6: Hematoxylin Eosin Staining for LMD
20.  
Deparaffinization and Rehydration1. Xylol20 seconds2. Xylol20 seconds3. Xylol20 seconds1. 100% Ethanol30 seconds2. 100% Ethanol30 seconds1. 95% Ethanol30 seconds2. 95% Ethanol30 seconds1. 70% Ethanol30 seconds2. 70% Ethanol30 secondsAqua dest. DEPC30 secondsFärbungHematoxylin (Harris, Merck)60 secondsAqua dest. DEPC30 secondsEosin Y Solution (Sigma)10 secondsDehydratisierungOH 70%30 secondsOH 95%30 secondsOH 100%30 secondsLet section air-dry uncovered~ 60 seconds
 
 

SECTION: Step 2: Tissue Processing
6. Cut samples immediatly after collection to an adequate size (no thicker than 1 cm) to allow formalin and paraffin penetration (and thus fixation) of the entire piece of tissue. 
SECTION: Step 2: Tissue Processing
7. Put sample into 4% neutral buffered formalin in an adequate sized vessel ensuring constant contact of tissue and formalin for 24-48 hours at room temperature.
SECTION: Step 3: Paraffin Embedding
9. Paraffin Embedding in Histocentre 2
SECTION: Step 3: Paraffin Embedding
8. Dehydration and Paraffinization in Citadel 1000

 

SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
12. Fasten cooled block into block holder of the rotary microtome.
SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
13. Cut 5 µm thick slides.

 

SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
11. Heat water bath with deionized water of the rotary microtome to 36°C.
SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
14. Mount unwrinkled sections on Superfrost Plus Langenbrick glass slides.
SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
15. Dry sections overnight at 37°C in heating cabinet (Heraeus Type 290).
SECTION: Step 4: Sectioning of Tissue Blocks with Microtome
16. Store slides at room temperature protected from light until further use. 

 

SECTION: Step 6: Hematoxylin Eosin Staining for LMD
19. 
Diethylpyrocarbonate (DEPC) treat alcohols and water used in this protocol and carry out final dehydrating steps with undenatured ethanol.
SECTION: Step 6: Hematoxylin Eosin Staining for LMD
19.1. 0.1% DEPC Solution:
999 ml deionized water + 1 ml DEPC 
Mix overnight
Autoclave in the morning
SECTION: Step 6: Hematoxylin Eosin Staining for LMD
18. Stain the sections according to a short hematoxylin eosin protocol to identify regions of interest for later LMD use.
SECTION: Step 6: Hematoxylin Eosin Staining for LMD
21. Let sections air-dry and do not put on a cover glass.
SECTION: Step 6: Hematoxylin Eosin Staining for LMD
21.1. Immediatly transfer stained slide to laser microscope or store in a sterile falcon tube at 4°C.
Do not freeze- as thawing causes water condensation on the slide which actives RNases.
SECTION: Step 7: Laser Microdissection and Scratch off
23. Encircle the area of interest with the freehand point to point function. 


SECTION: Step 7: Laser Microdissection and Scratch off
23.1. Calculate size of the area (µm2 * 5µm thickness) and expected RNA amount
SECTION: Step 7: Laser Microdissection and Scratch off
24. Set Power of the laser to the maximum to generate a marking that is macroscopically visible -> Laser Microdissection
  
 
   

SECTION: Step 7: Laser Microdissection and Scratch off
25. Transfer slide with laser microdissected sample to a workbench (which has been previously decontaminated with RNA Zap). 

  

SECTION: Step 7: Laser Microdissection and Scratch off
27.   
Prepare a 1.5 ml Eppendorf Tube with 150µl Proteinase K Buffer (PKD, supplied by RNeasy Kit) and 1µl Stabilizer Reagent. 
 


SECTION: Step 7: Laser Microdissection and Scratch off
26. Use a clean scalpel and spatula and scratch the marked area of interest off the slide.    


 


 
   
  

SECTION: Step 7: Laser Microdissection and Scratch off
29. At this point, the tube containing the collected LMD samples in buffer can be stored at 4°C for up to 24 hours
SECTION: Step 7: Laser Microdissection and Scratch off
28.  Transfer the collected scratched off sample into the tube containing buffer.
SECTION: Step 7: Laser Microdissection and Scratch off
30. Once enough tissue has been collected, freeze the sample at -20°C at least briefly and thaw it again right before the RNA extraction.
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
59. Use a Eppendorf Mastercycler for Reverse Transcription of up to 1µg RNA to cDNA in a 20µl volume with the BioRad iScript Synthesis Kit and protocol 
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
62.1. 4µl iScript primer mix of oligo dt and random primers. With this primer mix as many RNA fragments as possible can be  detected and transcribed
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
62.2. 1µl of Reverse Transcriptase
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
62.3. up to 15µl of RNA elute corresponding to 1µg of RNA 
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
62.4. add up to 20µl with Nuclease free water
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
63. Centrifuge 

SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
62. 
Use 0.2 Tube Stripes
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
64. Start BioRad protocol in Mastercycler 
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
64.4. Cool down and hold at 4°C
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
65. You receive 20µl of cDNA eluate with a concentration of 50 ng/µl
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
64.1. Priming step: 5 minutes at 25°C
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
64.2. Reverse transcription step: 20 minutes at 46°C
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
64.3. Inactivation of RT Step: 1 minute at 95°C

SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
66. Freeze at -20°C
 

SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
60. Set up a Sample-RT Control with RNA template and primer mix but no RT
SECTION: Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
61. Set up NTC control without RNA template but with RT and primer mix
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
31. Thaw Scratch Sample with PKD Buffer. 
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
32. Add 10µl of Proteinase K. Mix carefully. Do not vortex!
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
33. Incubate at 56°C in Eppendorf Thermomixer comfort (with shaking function) overnight. 



 

SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
36. Cool on ice for 3 min. 
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
34. Let sample rest at room temperature until heating block has reached 80°C
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
37. Centrifuge for 15 min at 20,000 x g (13,500 rpm).
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
35. Incubate sample at 80°C  for 15 minutes in Thermomixer
 

SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
38. Transfer the supernatant to a new 1.5 ml tube without disturbing the pellet.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
39. Add DNase Booster Buffer:
 approximately 16 μl = tenth of the total sample volume.

SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
41. Incubate at room temperature for 15 min. Flick tube from time to time (approx. every 3 minutes).
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
42. Add 320 μl Buffer RBC and mix thoroughly.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
43. Add 720 μl ethanol (100%)  and mix carefully but thoroughly. 
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
44. Transfer 650 μl of the sample to a supplied RNeasy MinElute spin column  in a 2 ml  tube. 
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
46. Discard the flowthrough.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
45. Centrifuge for 15 s at 8000 x g (10,000 rpm).
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
47. Transfer the rest of the sample to the same RNeasy spin column and centrifuge for another 15 s at 10 000 rpm. Discard the flowthrough.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
48. Add 500 μl Buffer RPE to the RNeasy MinElute spin column. Centrifuge for 15 s at 8000 x g (P10,000 rpm) to wash the spin column membrane. Discard the flowthrough.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
49. Add 500 μl Buffer RPE to the RNeasy MinElute spin column.  Close lid, centrifuge for 2 min at 8000 x g (10,000 rpm) to wash the spin column membrane.
Discard  the flowthrough and the collection tube.
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
50. Place the RNeasy MinElute spin column in a new 2 ml collection tube. Open the lid
of the spin column, and centrifuge at full speed for 5 min. Discard the collection tube with the
flow-through.
 

SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
51. Place the RNeasy MinElute spin column in a new 1.5 ml tube. Add
14–30 μl RNase-free water directly to the spin column membrane. Close the lid gently, and
centrifuge for 1 min at full speed to elute the RNA.

 

SECTION: Step 1: Sample Collection
1. During cesarean delivery: Collect sample of the uterine wall from the lower uterine segment (after delivery of the baby and before delivery of the placenta).

SECTION: Step 5: Gomori Trichrome Staining
17.   

 

SECTION: Step 11: qPCR (TaqMan techc)
68. Load wells:
Taqman Universal PCR Mastermix (2X)
Taqman Gene Expression Assay (10X)
cDNA (<100ng)
nuclease free water
SECTION: Step 11: qPCR (TaqMan techc)
69. Protocol:


 

SECTION: Step 11: qPCR (TaqMan techc)
67.1. Test samples in triplicates
SECTION: Step 11: qPCR (TaqMan techc)
67.2. Run interplate calibrator on each plate
 

SECTION: Step 11: qPCR (TaqMan techc)
67.3. Load plate according to sample maximization method: One gene per plate and maximum amount of samples
 

SECTION: Step 1: Sample Collection
2. Find scarred area in lower uterine segment (best visualized with intraoperative ultrasound when uterus is exposed just before childbirth. Mark the scarred area e.g. with cauter points. Place the uterotomy 1 cm above the identified scar).
SECTION: Step 1: Sample Collection
3. Cut out small piece of uterine tissue containing healthy myometrium and scarred area after child is born and before intravenous oxytocin application.
SECTION: Step 1: Sample Collection
4. Immediately continue with tissue processing.
SECTION: Step 12: Gel electrophoresis
71. Prepare Agarose gel (5%):

SECTION: Step 12: Gel electrophoresis
72. Electrophoresis
SECTION: Step 12: Gel electrophoresis
73. Analyze under UV light
SECTION: Step 12: Gel electrophoresis
70.1. Preparation of 1000 ml TAE (1X): 20 ml TAE (50X) + 980 ml H2O
SECTION: Step 12: Gel electrophoresis
71.2.  dissolve in microwave at 560W (boil until bubbles appear)

SECTION: Step 12: Gel electrophoresis
71.1. Weigh 5g agarose in 100 ml 1xTAE buffer and carefully swirl in the Erlenmeyer flask
SECTION: Step 12: Gel electrophoresis
71.3.  Let it cool down for approx. 10 minutes (swirl carefully) to 50-60 ° C

SECTION: Step 12: Gel electrophoresis
71.4. Solution should now be clear (otherwise boil again)

SECTION: Step 12: Gel electrophoresis
71.5. Add 10µl ethydium bromide (1µl per 10mL gel) to the cooled solution (do not boil)

SECTION: Step 12: Gel electrophoresis
71.6. Stir with a stir bar or by swirling -> liquid gel
SECTION: Step 12: Gel electrophoresis
71.7. Pour out the gel, make sure that there are no air bubbles in it (if necessary, use a pipette tip to remove it)
SECTION: Step 12: Gel electrophoresis
71.8. Insert the comb (0.5-1cm away from the upper edge)
SECTION: Step 12: Gel electrophoresis
71.9. Let it harden for approx. 1 hour until it is slightly milky → pour TAE Buffer (1X) over it → remove comb
SECTION: Step 12: Gel electrophoresis
72.1. Load the first pocket with 6µl of the dna ladder (marker) (should have the same volume as samples)

SECTION: Step 12: Gel electrophoresis
72.2. Load further pockets with 6µl mixture of 5µl PCR sample and 1µl loading buffer

SECTION: Step 12: Gel electrophoresis
72.3. Let it run for approx. 60 min at xxV (5 volts per cm of gel length)
SECTION: Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
40. Add 10 μl DNase I stock solution. Mix carefully. Do not vortex!
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54. Prepare gel
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55. Load chip
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
53.1. Heat in RNAse free Eppi for 2 minutes at 70°C
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
57. Measure in Bio-Analyzer
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
53.2. Aliquot and store at -80°C
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54.1. Centrifuge 550µl RNA-gel-matrix on spin filter for 10 minutes at 1500 rcf
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54.2. aliquot 65µl Gel and store at 4°C for up to four weeks

SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54.3. vortex blue RNA dye and centrifuge
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54.4. add 1µl RNA dye to 65µl gel

SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
54.5. vortex dye gel mix and centrifuge for 10 minutes at 13 000 rcf
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.1. Place the chip on the priming station
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.2. Apply 9µl of gel-dye mix to the G-well
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.3. Position the plunger at the level of 1mL and close the priming station
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.4. Press down the syringe until it is held by the clip →  wait 30s → then loosen the clip and hold for 5s (note: hold the syringe well as it moves back to its original position due to the negative pressure) → slowly draw back to 1mL
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.5. Open the priming station and apply 9 µl of gel-dye mix to each of the two G-wells with a white background
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.6. Apply 5µl of RNA marker (marked green) to all 12 sample wells and the ladder well
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.7. Apply 1µl of ladder to ladder well
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.8. Transfer 1µl of RNA sample to each of the 12 sample wells. Pipette 1 µl of RNA marker into free wells without samples
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.9. Vortex the chip at 2400 rpm for 1 min
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
57.1. Open the software 2100 Expert
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
58. Clean with H2O after measurement
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
56. Clean before use: 
- Electrode Cleaner "RNase ZAP": Pipette 350 µl RNase ZAP-water mixture (1:1) into a well, insert, close the flap, wait 1 minute and remove
- Electrode Cleaner "Water": Apply 350µl RNase-free water to a well, close the flap and wait 10s. Then take it out and let the water evaporate for another 10s with the flap open.
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
57.2. Select Eukaryote Total RNA Nano protocol
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
57.3. Insert the chip, enter the sample description in the table and start the measurement (takes about 30 minutes)
SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
52. 
 

SECTION: Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
55.10. Run the chip immediatly (within 5 minutes)