Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

The overall goal of this CDNA Library preparation is to enable successful sequencing of small RNAs that have been stored in formal and fixed paraffin-embedded tissues. This method is very useful for analyzing formal and fixed paraffin-embedded specimens. You extract RNA from up to 18 specimens, you analyze the content of all of these and obtain a profile of all of the microRNase for all the specimens.

To begin the protocol, add 8.5 microliters of previously-prepared ligation master mix to each of the 18 individually aliquoted FFPERNA samples. Gently flick the tubes, and centrifuge for two seconds. After centrifuging, store the tubes on ice.

Use a three microliter pipette to transfer one microliter of each adapter, to the corresponding FFPERNA samples containing RNA and ligation master mix. Do not pipette up and down, and change tips between FFPERNA samples. Close each tube, flick them to mix, and centrifuge them for two seconds.

Set the tubes on ice. Denature the reactions by placing the tubes on a heat block at 90 degrees celsius for one minute, and then immediately place them on ice. Next, use a three microliter pipette to transfer one microliter of the diluted ligation enzyme into each of the 18 RNA samples.

Set the ligations on ice, and place them in a four degrees celsius cold room for an overnight incubation of 18 hours. Place the tubes on a heat block for one minute at 90 degrees celsius to deactivate the ligation reaction and then return them to ice for at least two minutes to cool down. Transfer 1.2 microliters of the precipitation master mix into each tube.

Add 63 microliters of 100%ethanol. Close each tube, flick the tubes to mix, and centrifuge them for two seconds. Place the tubes on ice.

Combine the contents of all 18 tubes into a single 1.5 milliliter siliconized tube and invert three times to mix. Centrifuge the tubes for two seconds, and place them on ice for 60 minutes to precipitate. After precipitation, centrifuge the 1.5 milliliter tube with the pooled ligations.

Next, remove the supernatant with a one milliliter pipette tip but leave some liquid at the bottom. Tilt the tube, and use a 20 microliter pipette to remove the remaining supernatant. Vacuum-suction the supernatant with a Pasteur pipette with a 10 microliter tip, then resuspend the RNA pellet in 20 microliters of RNase-free water by flicking the tube.

Add 20 microliters of PAA gel loading solution to resuspend the RNA. Flick the tube, centrifuge the tube for two seconds at room temperature, and set the tube on ice. Replace the previously prepared 0.5 XTBE of the gel apparatus with fresh 0.5 XTBE, and load the latter, size markers, and ligated RNase in the center of the gel, leaving two empty wells on both sides.

Then run the gel. After running the gel, uncast the 15%page by removing one of the glass plates. Lightly spray the gel sitting on the glass plate with 10 microliters of fluorescent dye solution and 25 milliliters of 0.5 XTBE, and let it sit flat for five minutes in the dark.

Lay the glass with the gel on a blue-light transilluminator, and align both 19 nucleotide and 24 nucleotide size markers with a ruler to direct the excision of the ligated small RNAs. Then, excise the gel. Place the excised gel in a 0.5 milliliter tube designed to fragment gel slices, securely positioned into a 1.5 milliliter siliconized microcentrifuge tube.

Centrifuge the gel at 16 thousand times G for three minutes at room temperature. Resuspend the fragmented gel with 300 microliters of 400 millimolar sodium chloride solution. Close the tube, and seal it with paraffin film.

Agitate the contents on a thermo mixer at 11 hundred RPM in a four degrees celsius cold room for an overnight incubation. The next day, retrieve the tube from the thermo mixer and use a one milliliter pipette to transfer the solution onto a five micrometer filter tube, inserted into a 1.5 milliliter siliconized RNase-free collection tube. Centrifuge the tube for three minutes at 23 hundred times G at room temperature.

Discard the filter tube, add 950 microliters of 100%ethanol to the collection tube containing the filtered solution, and invert the tube to mix. Centrifuge the tube for two seconds, and place it on ice for 60 minutes. Then, precipitate the RNA pellet.

Next, dry the RNA pellet by opening the cap and carefully removing the supernatant with a one milliliter pipette, while leaving some liquid at the bottom. Use a 20 microliter pipette to remove all of the remaining supernatant without touching the pellet. Tilt the tube so any remaining solution moves to the opposite side of the pellet.

Use a Pasteur pipette to aspirate the supernatant and dry the pellet using a vacuum. Resuspend the RNA pellet in 5.6 microliters of RNase-free water. Defrost the reverse transcription reagents on ice for 15 minutes.

Set up a reverse transcription reaction by adding three microliters of 5X buffer, 4.2 microliters of 10X dioxynucleotide triphosphate, or DNTPs, and 1.5 microliter of dithiothreitol, or DTT. Gently flick the tube to mix the reaction, and centrifuge for two seconds to collect the solution. After the reaction is on a heat block at 90 degrees celsius for exactly 30 seconds, transfer it directly to a thermo mixer at 50 degrees celsius for two minutes to equalize the temperature.

Add 0.75 microliters of the reverse transcription enzyme directly into the solution. Flick gently to mix, and set the tube back on the thermo mixer at 50 degrees celsius for 30 minutes immediately. After 30 minutes, transfer the tube to a heat block at 95 degrees celsius for one minute to stop the reverse transcription.

Add 95 microliters of RNase-free water directly to the reverse transcription. Flick the tube to mix, and set it directly on ice for two minutes. Assemble the pilot PCR reaction and set up two PCR cycles on the thermo cycler.

Set up the pilot PCR reaction on the thermo cycler and start file one. At the end of file one, use a 20 microliter pipette to transfer 12 microliters from the pilot PCR reaction into 1.5 milliliter microcentrifuge tube containing three microliters of 5X gel loading dye. Mix by pipetting up and down.

And label it 10 cycles. Close the pilot PCR tube and start file two on the thermo cycler. At the end of file two, use a 20 microliter pipette to transfer 12 microliters of solution into a 1.5 milliliter microcentrifuge tube with three microliters of 5X gel loading dye, and label it 12 cycles.

Repeat the steps above to collect 12 microliters of PCR products from the pilot PCR reaction at PCR cycles 14, 16, 18, and 20. Then add three microliters of 5X gel loading dye to each of the 12 microliter PCR aliquots, and to the 20 nucleotide ladder containing three microliters of ladder and nine microliters of RNase-free water. After running the samples on an agarose gel with 0.5 XTBE, evaluate the gel on a UV box to identify the optimal PCR cycle.

Then set up the large-scale PCR reactions for agarose gel library purification. After PCR amplification, transfer nine microliters from each PCR tube into six 1.5 milliliter microcentrifuge tubes, containing three microliters of 5X gel loading dye. Prepare a 20 nucleotide ladder.

Load the ladder, negative PCR control, and six PCR products onto the agarose gel, and run the gel for 30 minutes at 120 volts. Verify the uniformity of the PCR implifications between lanes on a UV box, and take an image. Combine three PCR reactions into two 1.5 milliliter siliconized microcentrifuge tubes, add 27 microliters of five molar sodium chloride, and 950 microliters of 100%ethanol.

Invert the tubes to mix, and set them at 20 degrees celsius overnight to precipitate the PCR products. Using the protocol, the micro RNA expression correlation between the two fresh frozen tumor RNA and their specific FFPERNA specimen counterparts were ascertained. The plots between matched fresh-frozen and FFPE micro RNA indicate that the CDNA Library preparation provides a good reproducibility, as there is high correlation between the microRNA detected in specimens processed differently.

To determine if the optimized CNDA Library preparation protocol was applicable and reproducible with increasingly older FFPE specimens, micro RNA expression profiles from FFPE tissues archived for 18, 20, 22, 27, 30, and 35 years were obtained. The correlation coefficient remained above 0.96 regardless of the age of the specimen or the library preparation week. Therefore, the optimized CDNA Library preparation protocol provides a robust tool for reproducible analysis of FFPE specimens, regardless of their archival time.

So after watching this video you should have a good understanding on how to prepare your small RNA libraries and obtain a full microinexpression profile of all your precious formal and fixed paraffin-embedded specimens.