Extraction of total RNA and DNA from FFPE

A series of microsections were received from Dr Salvador Diaz-Cano (Department of Histopathology KCH). These sections had been cut from paraffin-embedded tissue blocks prepared from tumour tissue that had been surgically removed from patients with ACC or ACA. They were of thickness 0.7 mm and each of them was attached to a slide in the standard way. The first each of them had been stained and subjected to the histological evaluation by Dr Diaz-Cano, who designated areas of the tumour as central or peripheral.

Staining is known to affect the quality of extracted RNA and suitability for its application in further downstream steps. Following the labelling of the first section, further sections of waxed tissue were dissected using a scalpel blade into central and peripheral material and next transferred to two separate Eppendorf tubes. These pooled portions of material, derived from one patient, were then used for RNA extraction. Remaining sections were used for extraction of DNA, after dissection of the two zones as before. The number of sections needed to get sufficient RNA was first established (Section 3.3.5).

Materials and Methods

Figure 12. Workflow of Total RNA extraction from FFPE, using RNeasy FFPE Kit.

Extraction of total RNA from FFPE was carried out following the RNeasy FFPE Handbook using the RNeasy FFPE Kit. This kit is specifically designed to reverse as much as possible the chemical modifications of RNA caused by formaldehyde, such as crosslinking, which cannot be detected by standard ‘lab on-a-chip’ quality control. RNA originating from FFPE often has lower molecular weight in comparison to others. By isolating RNA molecules longer than 70 nucleotides, the kit provides recovery of RNA fragments, making them suitable for gene expression analysis.

Each sample was first dewaxed in the fume cupboard using 1 ml xylene. After thorough vortex mixing and centrifugation at maximum speed for 5 minutes, the xylene containing the paraffin was carefully poured off, taking care not to disturb the pellet remaining at the bottom, and then the dewaxing step was repeated. In the same way, each sample was then

His tologica l e va lua tion of tumour (s ta ining)

De pa ra ffiniza tion with xyle ne (x2)

S cra tching (pe riphe ra l a nd inte rna l to s e pa ra te tube s )

FFPE blo c k mic ro s e c tio ns

gDNA e limina tion with R Nas e-F ree D Nas e I a nd D Nas e B oos ter Buffe r

(incuba tion)

Adding of B uffer R P E (x2) Adding of B uffer R B C with e tha nol

a nd tra ns fe r to column S upe rna ta nt tra ns fe r to a ne w tube

Ce ntrifuga tion with the lid ope n in R Nas e-F ree Water

Wa s hing in e tha nol (x2)

Incuba tion with P roteinas e K a nd B uffer P K D

Elution with R Nas e-F ree Water

To tal RNA

Materials and Methods

washed with 96% aqueous ethanol. The pellet was left in the fume cupboard until completely dry.

Incubation with optimized lysis buffer containing Proteinase K was next carried out in order to release the RNA. A temperature gradient was applied, which allows the removal of chemical modifications of RNA, with a higher temperature incubation removing partial cross-linking. Modification of time and temperature improved the concentration and quality of the RNA (further described at “Optimization” – Section 3.3.5). Each sample was poured into 240 µl Buffer PKD. After vortexing, 10 µl Proteinase K solution was added. The sample was then mixed gently by pipetting and incubated overnight at 56⁰C, followed by 10 min incubation at 80⁰C the following day. After cooling on ice, the samples were centrifuged for 15 min at 13,500 rpm. The supernatants were transferred to new tubes.

DNA was removed by incubation with 10 µg Free DNase I in 25 µl DNase Booster Buffer for 15 min. Buffer RBC with ethanol was added to adjust binding conditions of RNA to the RNeasy MinElute Spin Column, while impurities were effectively washed from the column. Buffer RBC (500 µl) and ethanol (100 µl) were added to each sample. After thorough mixing, 700 µl was transferred to a column, followed by 30 s centrifugation at 9000 g. Buffer RPE (500 µl) was then layered onto the column membrane, followed by centrifugation for next 30 s. Centrifugation was repeated for 2 min. Changing a new collection tube under the column and placing the columns in the centrifuge and especially running with the lid open for 1 min were procedures aimed at ensuring that there would be no remaining contamination of total RNA.

For the last step, 17 µl RNase-Free Water was carefully poured onto the column membrane, followed by centrifugation at full speed for 3 minutes to elute the RNA.

Obtained RNA (2 µl) was immediately taken from the original tube and combined with 2 µl of RNase-Free Water in mini tubes and kept on ice ready for measurement of the concentration of total RNA.

Extraction of DNA from FFPE was carried out for the first samples using the QIAamp DNA FFPE Tissue Kit, according to the QIAamp DNA FFPE Tissue Handbook.

Thereafter, for the majority of samples, the REPLI-g FFPE Kit was used, following protocol taken from the REPLI-g Mini/Midi Handbook.

Purity and concentration of nucleic acids was measured using a Thermo Scientific

Materials and Methods