Optimized protocol for the RNA isolation of laser microdissected formalin-fixed paraffin-embedded uterine scar tissues for RNA expression analyses

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.

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.

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

Before cutting, cool tissue blocks at 4°C for 1 hour to harden the wax and thus allow easier cutting.

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.

Mount unwrinkled sections on Superfrost Plus Langenbrick glass slides.

Dry sections overnight at 37°C in heating cabinet (Heraeus Type 290).

Store slides at room temperature protected from light until further use.

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

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

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

Let sections air-dry and do not put on a cover glass.

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.

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

Transfer slide with laser microdissected sample to a workbench (which has been previously decontaminated with RNA Zap).

Use a clean scalpel and spatula and scratch the marked area of interest off the slide.

Prepare a 1.5 ml Eppendorf Tube with 150µl Proteinase K Buffer (PKD, supplied by RNeasy Kit) and 1µl Stabilizer Reagent.

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.

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.

Let sample rest at room temperature until heating block has reached 80°C

Incubate sample at 80°C for 15 minutes in Thermomixer

Cool on ice for 3 min.

Centrifuge for 15 min at 20,000 x g (13,500 rpm).

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.

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.

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

Run the chip immediatly (within 5 minutes)

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

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

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

Reverse transcription step: 20 minutes at 46°C

Inactivation of RT Step: 1 minute at 95°C

Cool down and hold at 4°C

You receive 20µl of cDNA eluate with a concentration of 50 ng/µl

Freeze at -20°C

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

Load plate according to sample maximization method: One gene per plate and maximum amount of samples

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

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