Feb 26, 2022
Optimized protocol for the RNA isolation of laser microdissected formalin-fixed paraffin-embedded uterine scar tissues for RNA expression analyses
- Alexander Paping1,
- Clara Basler1,
- Rebecca C. Rancourt1,
- Loreen Ehrlich1,
- Kerstin Melchior1,
- Wolfgang Henrich2,
- Thorsten Braun1
- 1Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Division of 'Experimental Obstetrics', Berlin, Germany;
- 2Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Obstetrics, Berlin, Germany
Protocol Citation: Alexander Paping, Clara Basler, Rebecca C. Rancourt, Loreen Ehrlich, Kerstin Melchior, Wolfgang Henrich, Thorsten Braun 2022. Optimized protocol for the RNA isolation of laser microdissected formalin-fixed paraffin-embedded uterine scar tissues for RNA expression analyses. protocols.io https://dx.doi.org/10.17504/protocols.io.byhgpt3w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 24, 2021
Last Modified: February 26, 2022
Protocol Integer ID: 53512
Keywords: qPCR, RNA isolation, RNA expression, LMD, LCM, laser microdissection, FFPE, uterine scar tissue
Funders Acknowledgement:
Deutsche Forschungsgemeinschaft Research Grants Programme
Grant ID: SCHW 1946/2-1
Deutsche Forschungsgemeinschaft Research Grants Programme
Grant ID: BR 2925/11-1
university research fund of Charité – Universitätsmedizin Berlin
Grant ID: 51517172–01
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
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.
Materials
Equipment
- Citadel 1000 (Shandon GmbH, Frankfurt Main, Germany)
- Histocentre2 (Shandon GmbH, Frankfurt Main, Germany)
- Microm HM 340E (Thermo Scientific, Walldorf, Germany)
- Heating Cabinet Heraeus Type 290 (Heraeus, Hanau, Deutschland)
- LMD5 (Leica Microsystems, Wetzlar, Germany)
- Thermomixer comfort (Eppendorf, Hamburg, Germany)
- Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA)
- Eppendorf Mastercycler epgradient S (Eppendorf, Hamburg, Germany)
- ABI 7500 (Applied Biosystems,ThermoFisher Scientific, Waltham, MA, USA)
Materials/Reagents
- glass slides (SuperFrost Plus; R. Langenbrinck, Emmendingen, Germany)
- sterile scalpel (..)
- RNase inhibitor (Sigma Aldrich, Merck, Darmstadt, Germany)
- RNeasy FFPE Kit (Qiagen, Hilden, Germany)
- RNA 6000 Nano Kit (Agilent technologies, Santa Clara, CA, USA)
- iScript cDNA Synthesis Kit (BioRad, Feldkirchen, Germany)
- Taqman probe technology (ThermoFisher Scientific, Waltham, MA USA)
- Universal PCR Mastermix (ThermoFisher Scientific, Waltham, MA USA)
- TAE Buffer 50% (Rotiphorese, Roth, Weiden, Germany)
Step 1: Sample Collection
Step 1: Sample Collection
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).
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).
Cut out small piece of uterine tissue containing healthy myometrium and scarred area after child is born and before intravenous oxytocin application.
Immediately continue with tissue processing.
Step 2: Tissue Processing
Step 2: Tissue Processing
Treat surfaces and eqipment with RNase Away to prevent RNA degradation by RNases during tissue preparation.
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.
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.
Step 3: Paraffin Embedding
Step 3: Paraffin Embedding
Dehydration and Paraffinization in Citadel 1000
| A | B | |
| 70% Ethanol | 3 hours | |
| 70% Ethanol | 1 hour | |
| 70% Ethanol | 1 hour | |
| 80% Ethanol | 1 hour | |
| 96% Ethanol | 1 hour | |
| 100% Ethanol | 1 hour | |
| 100% Ethanol | 1 hour | |
| 100% Ethanol | 1 hour | |
| Chloroform | 1 hour | |
| Chloroform | 2 hours | |
| Paraffin | 2 hours | |
| Paraffin | 3 hours |
Paraffin Embedding in Histocentre 2
Step 4: Sectioning of Tissue Blocks with Microtome
Step 4: Sectioning of Tissue Blocks with Microtome
Before cutting, cool tissue blocks at 4°C for 1 hour to harden the wax and thus allow easier cutting.
1h
Heat water bath with deionized water of the rotary microtome to 36°C.
Fasten cooled block into block holder of the rotary microtome.
Cut 5 µm thick slides.
Note
Beforehand, you may want to trim the block carefully with 20µm thickness to ensure correct positioning and to expose enough surface of the tissue block to cut a representative section of the entire specimen.
Mount unwrinkled sections on Superfrost Plus Langenbrick glass slides.
Dry sections overnight at 37°C in heating cabinet (Heraeus Type 290).
12h
Store slides at room temperature protected from light until further use.
Note
Sections should be safe and protected from RNases in Paraffin therefore storage at room temperature should be fine.
If you have enough space you can store blocks and slides at 4°C.
Step 5: Gomori Trichrome Staining
Step 5: Gomori Trichrome Staining
| A | B | C | |
| Deparaffinization and Rehydration | |||
| Xylol | 5 min | ||
| Xylol | 5 min | ||
| 96% Ethanol | 2 min | ||
| 70% Ethanol | 2 min | ||
| 50% Ethanol | 2 min | ||
| Aqua dest. | 2 min | ||
| Staining | |||
| Boin Solution | 30 min | 56°C | |
| Running tab water | 5 min | ||
| Weigert's hematoxylin | 10 min | ||
| Running tab water | 10 min | ||
| Trichrome Solution | 25 min | ||
| 0.5% acetic acid | 2 min | ||
| 0.5% acetic acid | 1 min | ||
| Dehydration | |||
| 96% Ethanol | 1 min | ||
| 1. 100% Ethanol | 2 min | ||
| 2. 100% Ethanol | 2 min | ||
| Xylol | 5 min | ||
| Xylol | 5 min | ||
| Put on cover glass | |||
Step 6: Hematoxylin Eosin Staining for LMD
Step 6: Hematoxylin Eosin Staining for LMD
8m
8m
Stain the sections according to a short hematoxylin eosin protocol to identify regions of interest for later LMD use.
Diethylpyrocarbonate (DEPC) treat alcohols and water used in this protocol and carry out final dehydrating steps with undenatured ethanol.
0.1% DEPC Solution:
999 ml deionized water + 1 ml DEPC
Mix overnight
Autoclave in the morning
12h
| A | B | |
| Deparaffinization and Rehydration | ||
| 1. Xylol | 20 seconds | |
| 2. Xylol | 20 seconds | |
| 3. Xylol | 20 seconds | |
| 1. 100% Ethanol | 30 seconds | |
| 2. 100% Ethanol | 30 seconds | |
| 1. 95% Ethanol | 30 seconds | |
| 2. 95% Ethanol | 30 seconds | |
| 1. 70% Ethanol | 30 seconds | |
| 2. 70% Ethanol | 30 seconds | |
| Aqua dest. DEPC | 30 seconds | |
| Staining | ||
| Hematoxylin (Harris, Merck) | 60 seconds | |
| Aqua dest. DEPC | 30 seconds | |
| Eosin Y Solution (Sigma) | 10 seconds | |
| Dehydration | ||
| 70% Ethanol | 30 seconds | |
| 95% Ethanol | 30 seconds | |
| 100% Ethanol | 30 seconds | |
Note
Be careful to get rid of all remaining paraffin before RNA extraction as cells still embedded in the latter cannot be isolated. This is especially important when using sections thicker than 5µm.
8m
Let sections air-dry and do not put on a cover glass.
1m
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.
Step 7: Laser Microdissection and Scratch off
Step 7: Laser Microdissection and Scratch off
Put slide into the slide holder with the specimen facing downwards.
Encircle the area of interest with the freehand point to point function.
Calculate size of the area (µm2 * 5µm thickness) and expected RNA amount
Set Power of the laser to the maximum to generate a marking that is macroscopically visible -> Laser Microdissection
Gomori-Trichrome stained sample after Image J analysis
Hematoxylin Eosin stained sample with scar region outlined by laser,
for demonstrational purpose repainted with GIMP
Transfer slide with laser microdissected sample to a workbench (which has been previously decontaminated with RNA Zap).
Preparation for scratch off:
- 1,5 ml tube with Stabilizer reagent, Proteinase K buffer
- two sample slides, one stained with Hematoxylin Eosin, one with Gomori Trichrome
- clean scalpel and spatula
Use a clean scalpel and spatula and scratch the marked area of interest off the slide.
Note
You may decontaminate your tools with UV light and/or RNAZap before scratch off
Exemplary Hematoxylin Eosin stained samples after laser microdissection and scratch off
Prepare a 1.5 ml Eppendorf Tube with 150µl Proteinase K Buffer (PKD, supplied by RNeasy Kit) and 1µl Stabilizer Reagent.
Note
Use 20µl of Stabilyzer Reagent per 1mg of tissue.
Transfer the collected scratched off sample into the tube containing buffer.
At this point, the tube containing the collected LMD samples in buffer can be stored at 4°C for up to 24 hours
Once enough tissue has been collected, freeze the sample at -20°C at least briefly and thaw it again right before the RNA extraction.
Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
Step 8: RNA Extraction (Adapted Qiagen FFPE RNeasy Kit Protocol)
Thaw Scratch Sample with PKD Buffer.
Add 10µl of Proteinase K. Mix carefully. Do not vortex!
Incubate at 56°C in Eppendorf Thermomixer comfort (with shaking function) overnight.
Note
Incubation for digestion of proteins, reversal of RNA crosslinks for optimal RNA performance
Qiagen protocol says 10 minutes but this has not proven sufficient. Might need optimization up to 24 hours incubation time.
12h
Let sample rest at room temperature until heating block has reached 80°C
Incubate sample at 80°C for 15 minutes in Thermomixer
Note
The incubation at 80°C in Buffer PKD partially reverses formaldehyde modification of nucleic
acids. Longer incubation times or higher incubation temperatures may result in more
fragmented RNA, but may also result in slightly lower CT values in downstream applications,
such as real-time RT-PCR.
15m
Cool on ice for 3 min.
3m
Centrifuge for 15 min at 20,000 x g (13,500 rpm).
15m
Transfer the supernatant to a new 1.5 ml tube without disturbing the pellet.
Add DNase Booster Buffer:
approximately 16 μl = tenth of the total sample volume.
Add 10 μl DNase I stock solution. Mix carefully. Do not vortex!
Incubate at room temperature for 15 min. Flick tube from time to time (approx. every 3 minutes).
Add 320 μl Buffer RBC and mix thoroughly.
Add 720 μl ethanol (100%) and mix carefully but thoroughly.
Transfer 650 μl of the sample to a supplied RNeasy MinElute spin column in a 2 ml tube.
Centrifuge for 15 s at 8000 x g (10,000 rpm).
Discard the flowthrough.
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.
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.
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.
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.
Note
Qiagen: To avoid damage to their lids, place the spin columns into the centrifuge with at least one
empty position between columns. Orient the lids so that they point in a direction opposite to
the rotation of the rotor.
It is important to dry the spin column membrane, since residual ethanol may interfere with
downstream reactions.
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.
Note
Elution with smaller volumes of RNase-free water leads to higher total RNA concentrations, but
lower RNA yields.
The dead volume of the RNeasy MinElute spin column is 2 μl: elution with 14 μl RNase-free
water results in a 12 μl eluate.
Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
Step 9: Control of RNA Integrity (Agilent 2100 Bioanalyzer, Nano Kit)
Note
Use RNA 6000 Nano kit for RNA concentration of 25 to 500 ng/μL
Use RNA 6000 Pico kit for lower concentrations like 50 to 5 000 pg/μL
Prepare Ladder
Heat in RNAse free Eppi for 2 minutes at 70°C
Aliquot and store at -80°C
Prepare gel
Centrifuge 550µl RNA-gel-matrix on spin filter for 10 minutes at 1500 rcf
aliquot 65µl Gel and store at 4°C for up to four weeks
vortex blue RNA dye and centrifuge
add 1µl RNA dye to 65µl gel
vortex dye gel mix and centrifuge for 10 minutes at 13 000 rcf
Load chip
Place the chip on the priming station
Apply 9µl of gel-dye mix to the G-well
Position the plunger at the level of 1mL and close the priming station
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
Open the priming station and apply 9 µl of gel-dye mix to each of the two G-wells with a white background
Apply 5µl of RNA marker (marked green) to all 12 sample wells and the ladder well
Apply 1µl of ladder to ladder well
Transfer 1µl of RNA sample to each of the 12 sample wells. Pipette 1 µl of RNA marker into free wells without samples
Vortex the chip at 2400 rpm for 1 min
Run the chip immediatly (within 5 minutes)
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.
Measure in Bio-Analyzer
Open the software 2100 Expert
Select Eukaryote Total RNA Nano protocol
Insert the chip, enter the sample description in the table and start the measurement (takes about 30 minutes)
Clean with H2O after measurement
Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
Step 10: cDNA Synthesis (cDNA synthesis Kit BioRad protocol)
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
Set up a Sample-RT Control with RNA template and primer mix but no RT
Set up NTC control without RNA template but with RT and primer mix
Use 0.2 Tube Stripes
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
1µl of Reverse Transcriptase
up to 15µl of RNA elute corresponding to 1µg of RNA
add up to 20µl with Nuclease free water
Centrifuge
Start BioRad protocol in Mastercycler
Priming step: 5 minutes at 25°C
5m
Reverse transcription step: 20 minutes at 46°C
20m
Inactivation of RT Step: 1 minute at 95°C
1m
Cool down and hold at 4°C
You receive 20µl of cDNA eluate with a concentration of 50 ng/µl
Freeze at -20°C
Note
Before freezing, aliquoting might be useful. Adjust concentration and amount to needs before freezing as multiple freeze-thaw-cycles result in higher Ct values.
Step 11: qPCR (TaqMan techc)
Step 11: qPCR (TaqMan techc)
Carry out qPCR in Singleplex using Taqman probe technology in 96 well plates in an ABI 7500 (Applied Biosystems)
Test samples in triplicates
Run interplate calibrator on each plate
Note
This can be a mix of cDNA or gDNA run with one of your housekeeping genes that should be the same on every plate. This way the factor of interplate variances can be detected and included in later analyses.
Load plate according to sample maximization method: One gene per plate and maximum amount of samples
Note
If you are analyzing multiple groups, samples of each groups should be mixed on plate, so that every group is represented on every plate
Load wells:
- Taqman Universal PCR Mastermix (2X)
- Taqman Gene Expression Assay (10X)
- cDNA (<100ng)
- nuclease free water
Protocol:
| A | B | C | D | |
| enzyme activation | 10 min | 95°C | ||
| denaturation | 15 sec | 95°C | 45 cycles | |
| annealing + elongation | 1 min | 60°C |
Step 12: Gel electrophoresis
Step 12: Gel electrophoresis
Prepare TAE Buffer (1X):
Preparation of 1000 ml TAE (1X): 20 ml TAE (50X) + 980 ml H2O
Prepare Agarose gel (5%):
Weigh 5g agarose in 100 ml 1xTAE buffer and carefully swirl in the Erlenmeyer flask
dissolve in microwave at 560W (boil until bubbles appear)
Let it cool down for approx. 10 minutes (swirl carefully) to 50-60 ° C
Solution should now be clear (otherwise boil again)
Add 10µl ethydium bromide (1µl per 10mL gel) to the cooled solution (do not boil)
Stir with a stir bar or by swirling -> liquid gel
Pour out the gel, make sure that there are no air bubbles in it (if necessary, use a pipette tip to remove it)
Insert the comb (0.5-1cm away from the upper edge)
Let it harden for approx. 1 hour until it is slightly milky → pour TAE Buffer (1X) over it → remove comb
Electrophoresis
Load the first pocket with 6µl of the dna ladder (marker) (should have the same volume as samples)
Load further pockets with 6µl mixture of 5µl PCR sample and 1µl loading buffer
Let it run for approx. 60 min at xxV (5 volts per cm of gel length)
Analyze under UV light