RNA Extraction from Decalcified and Non-decalcified Formalin-Fixed Paraffin-Embedded Tissues

Abstract

Bone tumors are very rare. They account for about 0.8% of all human neoplasms. Molecular techniques have a quite limited application in this field, because few entities show characteristic aberrations useful for diagnostic and therapeutical purposes. The prototype of these entities is the Ewing sarcoma, typically recognized by recurrent translocations. Often are described new aberrations, such as point mutations or gene amplifications that can occur in bone tumors. The major problem in this very specialized field is the kind of tissue, the bone, that needs decalcification, and this treatment can interfere with nucleic acid isolation. In the decalcified tissues, the affordability of molecular techniques is low (reported to be around 20%). For this reason, it is important to sample bone tumors before the decalcification process in order to store adequate tissue in biobanks for molecular purposes. A valid alternative choice could be to use adequate systems of decalcification that sufficiently preserve the quality of nucleic acids.

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1 Introduction and Purpose

This protocol provides a method for obtaining RNA suitable for RT-PCR analysis from decalcified and ­non-decalcified formalin-fixed and paraffin-embedded (FFPE) tissue specimens. Decalcification causes RNA fragmentation and it is generally believed that this damage precludes successful RT-PCR. For the best performance in calcified tissue, it is necessary to choose an appropriate protocol to decalcify the tissue in order to perform histological, immunological, and molecular studies. The protocol below describes the decalcification procedure using formic acid and nitric acid [1]. For decalcified and non-decalcified FFPE tissue samples, we have standardized an RNA extraction protocol based on a commercial kit (i.e., Pinpoint slide RNA isolation systemII, Zymo Research Corp) [2–4]. This kit for nucleic acid isolation is specifically developed for archive tissues. Some modifications have been made in order to obtain good-quality RNA, depending on the type, volume, and treatment of the tissue [5].

The RNA extraction procedure is based mainly on deparaffinization, tissue isolation, and proteolytic digestion with Proteinase K. The time required for the entire procedure is approximately 6 h.

2 Protocol

2.1 Reagents

Listed below are the products we use, but similar reagents from other manufacturers may be selected:

• Nitric acid 65% solution (Fluka or Sigma-Aldrich)

• Formic acid 98% solution (Fluka or Sigma-Aldrich)

• Xylene (Fluka or Sigma-Aldrich)

• Absolute, 96% and 70% ethanol (Fluka or Sigma-Aldrich)

• 20 mg/ml Proteinase K (stock solution): (Zymo Research Corp). Dissolve 5 mg of Proteinase K in 250 μl of storage glycerol Buffer. Store at −20°C.¹

• RNAse Away ² (Invitrogen) ready-to-use solution for eliminating RNase and DNA contamination from labware

• Sterile H ₂ O

2.2 Equipment

• Disinfected adjustable pipettes,³ range: 0.1–2 μl, 2–10 μl, 20–200 μl, 100–1,000 μl

• Nuclease-free aerosol-resistant pipette tips

• 1.5 ml tubes (autoclaved)

• Sterile or disposable tweezers

• Microtome, with new blade

• Orbital shaker

• Refrigerated MicroCentrifuge suitable for centrifugation of 1.5 ml tubes

• Waterbath

• Spectrophotometer

• Clean Micro slides with ground edges (76  ×  26 mm)

• Glass bowl

• Rack for slides

2.3 Method

2.3.1 Bone Fixation and Bone Decalcification Protocol

• For a large bone specimen, cut the sample in a large coronal or axial section using the band saw.⁴

• Fix the calcified sections in 4% neutral buffered formalin⁵ at room temperature for 24–48 h (for biopsy specimens the time of fixation is approximately 6 h)

• Submerge the samples in the decalcifying solution⁶ at room temperature for 7–10 days depending on size and calcification level (for biopsy specimens, the decalcification time is about 1–3 h)

• Wash the samples in tap water to remove the decalcifying solution.

• Dissect the specimen into smaller samples, then proceed with routine paraffin-embedding tissue protocol.

2.3.2 Sample Preparation

• If possible, cool the paraffin blocks at −20°C or on ice in order to facilitate cutting of the sections.⁷

• Using a clean, sharp microtome blade,⁸ cut two to five sections of 6–8 μm thickness depending on the size of the sample. Discard the first section and displace the other sections on a floating-out bath of clean deionized water at 40°C.

• Mount the sections on clean glass slides using sterile tweezers.

• Dry the sections at 60°C for 10 min.

• The first sections are used for RNA extraction; while the last section is hematoxylin-eosin-stained and used to check the morphology in order to select the area with adequate cellularity and to compare it with the unstained sections.

• Mark the selected area of interest for RNA isolation with a diamond tip.

2.3.3 Deparaffinization

• Submerge the slides in clean xylene at room temperature for 1 h changing the xylene once after approximately 30 min⁹.

• Hydrate the slides by washing progressively for 2 min in clean ethanol 100%, 96%, 75%, and then in pure water¹⁰.

• When the tissue has been decalcified, incubate the hydrated slides in 0.001N EDTA pH 8.0 for 40 min in orbital shaker at room temperature.

• Wash the slides in distilled water for 5 min. Repeat this step.

• Air dry the sections, leaving the slide at room temperature for approximately 10 min or under a hood for 2–3 min.

2.3.4 Tissue Isolation and Digestion

• Apply the Pinpoint solution to the selected area on the slide to remove the tissue region. The amount of solution depends on the tissue area¹¹

• Allow the Pinpoint solution to dry completely at room temperature.¹² When it is dry, it appears as a blue film embedding the tissue and cells underneath.

• Remove the embedded tissue from the slide¹³ and transfer to a sterile tube.

• Centrifuge briefly to locate the tissue sample at the bottom of the tube

• Add to the recovered tissue, 25 μl of digestion buffer 1× supplemented with Proteinase K at a final concentration of 1 mg/ml and mix ­gently. The amount of digestion buffer depends on the amount of tissue. The digestion buffer must cover the tissue pellet completely.

• Incubate the tube in a waterbath at 55°C for 4 h¹⁴.

• Centrifuge the tube briefly at the end of incubation.

2.3.5 RNA Extraction

• Add 50 μl of RNA extraction Buffer and mix well with a micropipette.

• Add 75 μl of 100% ethanol to the tube. Vortex lightly.

• Transfer the mixture to a Column in a 2 ml collection tube¹⁵

• Spin the column at 10,000 rpm in a microcentrifuge for 1 min.

• Add 200 μl of RNA wash buffer to the column and centrifuge at 10,000 rpm for 1 min. Repeat this step.

• Transfer the column into a new RNase-free 1.5 ml tube.

• Add 10 μl of distilled water directly to the membrane. Wait for 2 min.

• Spin the column at 10,000 rpm in a microcentrifuge for 1 min to elute the RNA¹⁶

• For RNA measurement, pipette 121 μl of sterile water into a fresh tube and add 4 μl of RNA extract (dilution factor = 25). Determine the RNA concentration photometrically at 260 and 280 nm (see Chap. 16, Sect. 16.2.1).¹⁷

2.4 Troubleshooting

• RNA degradation. RNA is highly susceptible to RNase digestion, we encourage the use of freshly prepared sections. If a sample cannot be processed immediately, store it at ≤70°C or submerge it in 96% ethanol at −20°C. Processing of tissue sections stored for 1 month or more at room temperature is not recommended.

• Insufficient RNA. Make sure an appropriate sampling area is selected for processing. Select an area of the tissue that will contain ≥50 cells. Increase the sampling area if the tissue type contains few cells (e.g., fatty tissue, fibrous or cartilaginous tissue). The sampling size can vary from 1 mm² to over 100 mm².

• DNA contamination. Traces of fragmented DNA may be present in the eluted RNA fraction. DNA-free RNA can be obtained with subsequent DNase I treatment.