Why do we do RNA extraction?

4.2 RNA isolation

The RNA extraction was performed as previously described (Nercessian et al., 2005) with the following modifications. Microcosm samples (0.5 g) were resuspended in 0.75 ml of RNA extraction buffer (0.15 mM NaH2PO4/Na2HPO4 buffer, pH 7.5; 5% CTAB, 1 M NaCl, 2% SDS; and 2% N-lauroylsarcosine sodium salt). The DNase I treatment was carried out using the DNAfree kit (BioLabs, Ambion) in accordance with the manufacturer's instructions. The RNA samples were further purified using the RNeasy columns (Invitrogen, USA). An additional DNase I treatment was carried out directly on the RNeasy columns using the RNase-Free DNase Set (QIAGEN) in accordance with manufacturer's instructions. The integrity of the RNA preparations was tested on a Bioanalyzer 2100 (Agilent), using Agilent RNA 6000 nano-kit, as suggested by the manufacturer.

Rationale

During RNA extraction, the most important step is the inactivation of endogenous RNase. Therefore, many protocols of RNA extraction include the usage of strong denaturants as guanidium salt, which disrupts the cells, solubilizes its components, and denature the endogenous RNase at the same time. The basis of the method is phase separation on the centrifugation of a mixture of aqueous sample and solution of phenol and chloroform. This results in the upper aqueous phase containing nucleic acid and the lower organic phase have protein. The acidic pH provided by sodium acetate causes the DNA to be organic while RNA at the aqueous phase. In neutral condition both RNA and DNA partitioned in the aqueous phase. Guanidinium cyanate aids in the denaturation of proteins (including RNase) and β-mercaptoethanol, and sarcosine too acts as denaturing agents. Then isopropanol (added to the aqueous phase) causes the RNA to precipitate out.

Northern Blots

RNA Extraction. Approximately 30 million cells are collected in a sterile 50-ml tube (Falcon type) for each extraction and pelleted by centrifugation (3000 g, 5 min, 4°). The pellet is immediately resuspended in 1 ml of TRIzol reagent (GIBCO-BRL, Gaithersburg, MD) or TRI reagent (Sigma, St. Louis, MO). Polysaccharides, membranes, and unlysed cells are eliminated by centrifugation (12,000 g, 10 min, 4°). At this step the supernatant is either stored at – 80° or immediately treated according to the supplier protocol. Briefly, add 200 µl of chloroform, incubate at room temperature for 2 min, and centrifuge (12,000 g, 15 min, 4°). The supernatant (600 µl emented with 500 µl of 2-propanol and centrifuged (12,000 g, 10 min, 4°) after a 10-min incubation at room temperature. The RNA pellet is washed [1 ml of 70%(v/v) ethanol and centrifugation at 8000 g for 10 min at 4°], dried at room temperature for 30 min, and resuspended in 20 µl of Milli-Q (Millipore, Bedford, MA)-treated sterile water. Total RNA extracts can be stored at − 20° for at least 1 month.

Gel Electrophoresis and Blotting. Before use all electrophoretic equipment is treated with 0.1 N NaOH to eliminate RNases. A 1.5% (w/v) agarose gel is prepared in 1 × MOPS buffer [20 mM morpholine propane sulfonic acid (MOPS), 30 mM sodium acetate, 10 mM Na2EDTA, pH 7], After melting of the agarose in a microwave oven, formaldehyde is added (20%, v/v) and the gel is poured. For each sample the RNA concentration is determined by spectrophotometry and a volume corresponding to 10 µg is complemented with Milli-Q sterile water to a final volume of 8 µl. Each sample is supplemented with 14 µl of formamide, 5 µl of formaldehyde, and 3 µl of 10 x MOPS buffer, denatured at 70° for 10 min, and supplemented with 3 µl of sterile loading buffer [0.25% (w/v) bromphenol blue, 30% (v/v) glycerol]. The samples are then loaded onto the gel. When necessary, 8 µl of RNA ladder (RNA Ladder; GIBCO-BRL) is also loaded. Migration is performed in 1 × MOPS buffer at a constant 100 V for 3 hr.

After migration, the gel can be stained with ethidium bromide (1.25 µg/ml, 5 min). The gel is then destained by several 30-min washes in sterile water. Washes are stopped when ribosomal RNAs and ladder bands can be visualized under UV.

The RNAs are blotted onto a positive membrane (Appligene; Qbiogene, Carlsbad, CA) by capillarity in 2× SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 16 hr. The membrane is rinsed in distilled water for 5 min and the RNAs are covalently fixed onto the membrane by UV cross-linking (UV Stratalinker 1800; Stratagene, La Jolla, CA).

Labeling of Probes. The probes are synthesized by random priming (Non-aprimer kit II; Appligene, Qbiogene). The constitutive probe is a fragment of the coding region of a G protein β subunit-like polypeptide.13 Because TRXs belong to a multigene family it is important to avoid cross-hybridization between different TRXs. For this purpose, the TRX probes correspond to the 3′ untranslated region of thioredoxin h and m isoforms,14 obtained by polymerase chain reaction (PCR). The Fd probe, used as a control, also corresponds to the 3′ untranslated region of the gene.

Hybridization and Washing. The membranes are prehybridized for 2 hr and hybridized overnight in 0.5 M sodium phosphate buffer (pH 7.2), 1 mM Na2EDTA, 7% (w/v) sodium dodecyl sulfate (SDS), and 1% (w/v) bovine serum albumin (BSA). Hybridization is performed at 62° for all probes. Washes are performed in 40 mM sodium phosphate buffer (pH 7.2), 1 mM Na2EDTA, 1% (w/v) SDS at ambient temperature (twice, 5 min each) and the final wash is performed at 62° for 20 min in the same buffer. Several exposure times are used in order to be in the linear response range of the film (X-Omat; Kodak, Rochester, NY).

Membrane Stripping. After hybridization the blot can be stripped by two rounds of boiling in 0.01× SSC, 0.5% (w/v) SDS and then hybridized with another probe. This stripping can be performed four or five times without altering the signals.

7.2 Buffers and solutions

Cytoplasmic RNA extraction buffer: 50 mM Tris–Cl, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.5% (v/v) NP-40. Prepare with DEPC-treated water and filter sterilize. Add ribonucleoside-vanadyl complex (New England Biolabs) to a final concentration of 10 mM just prior to use.

5× RNase H buffer: 100 mM HEPES-KOH, pH 8.0, 250 mM KCl, 20 mM MgCl2

10× T4 RNA ligase buffer: 330 mM Tris acetate (pH 7.8), 660 mM potassium acetate, 100 mM magnesium acetate, 5 mM dithiothreitol

10× BPTE electrophoresis buffer: 100 mM PIPES, 300 mM Bis–Tris, 10 mM EDTA (pH 8.0). The final pH of the 10× buffer is ≈6.5.

2× Glyoxal denaturation buffer: 1.125 M deionized glyoxal, 60% dimethyl sulfoxide, 1× BPTE buffer, 0.02% bromophenol blue, 0.02% xylene cyanol FF, 0.02 mg/ml ethidium bromide

Formamide loading buffer: 95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 18 mM EDTA, 0.025% SDS

20× SSC: 3 M NaCl, 0.3 M sodium citrate

16.1 Materials/Equipment

For RNA extraction:1.

Cell culture(s)

RNA extraction kit

RNase-free or DEPC- (diethylpyrocarbonate) treated water

RNase-free pipette tips (these can be purchased gamma irradiated and certified free of RNases and DNases, for example the BioClean range from Rainin) and plasticware

For microarray analysis:

Materials as detailed in the online documentation for microarrays:

Ambion ® WT Expression Kit (Applied Biosystems) manual

GeneChip ® WT Terminal Labeling and Hybridization User Manual (Affymetrix ®)

GeneChip ® Expression Wash, Stain and Scan User Manual (Affymetrix ® ), materials are available to purchase from the Affymetrix website (http://www.affymetrix.com)

For FISH:1.

Cell culture(s)

Glass slides or 2 mm thick polycarbonate substrates

Coverslips 22 mm

Humidified chamber

Parafilm

Waterbath or oven that can reach 80 °C

Basal media (recipe shown suitable for the culture of MSCs): αMEM (PAA Laboratories, cat. no. 11140-035) 500 ml bottle supplemented with 10% foetal bovine serum (FBS) (SIGMA, cat. no. F9665), 1% (v/v) 200 mM l-glutamine (SIGMA, cat. no. G7513) and antibiotics (6.74 U/ml penicillin/streptomycin (SIGMA, cat. no. P0781) and 0.2 mg/ml Fungizone, Gibco, cat. no. 15290-026)

HEPES saline: 5.4 mM KCl, 0.14 M NaCl, 5.5 mM d-Glucose, 10 mM HEPES, 0.01% (v/v) phenol red, pH 7.5

FISH fixative: 3:1 methanol:acetic acid

20 × SSC (saline sodium citrate) stock solution (autoclaved): 3 M NaCl, 0.3 M tri-sodium citrate, pH 7.4

1 × SSC buffer: dilute from 20 × SSC stock using double-distilled water

4 × SSC buffer: dilute from 20 × SSC stock using double-distilled water

Biotinylated human chromosome 1 paint (or other chromosome of interest) (Cambio, Cambridge, UK)

100%, 70%, 90% (v/v) ethanol (C2H5OH). Dilutions of ethanol to be made with autoclaved double-distilled water

Denaturation solution: 7:1 formamide: 2 × SSC buffer (from the diluted 20 × SSC stock)

StarFISH Biotin Painting Kit with Fluorescein isothiocyanate (FITC) (Cambio, cat. no. 1089-KT-50).

Image J (Rasband, W.S., Image J, U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/)

Prism (GraphPad, available to purchase from http://www.graphpad.com/prism)

6.2.1 Total RNA extraction

Timing: 4 + 16 h incubation at − 80 °C.

Total RNA extraction for 3′-mRNA-Seq is shown on the upper panel of Fig. 3A.

Cell lysis

Remove RPMI from the macrophage culture

Wash with ice-cold PBS

Add 250 μL Trizol per 6-multiwell and pipet up and down to homogenize

Note: Trizol contains acidic phenol to separate RNA from the remaining fractions, and guanidinium thiocyanate to inactivate endogenous RNases.

Transfer to RNase-free microcentrifuge tube

Incubate samples at RT for 2 min to dissociate the nucleoprotein complex

Add 200 μL of chloroform per each mL Trizol reagent

Mix them thoroughly by inverting the tube

Incubate at RT for 1 min

Centrifuge at 4 °C for 15 min at 12000 g

Transfer the upper aqueous phase to a new 1.5 mL RNase-free microcentrifuge tube

Critical: avoid taking out the organic layer and lower phase containing contaminants (phenol from Trizol, DNA, and protein).

RNA precipitation

Add an equal volume of isopropanol to the samples. Add 1 μL of glycogen to act as co-precipitant

Vortex briefly

Incubate at − 80 °C for 16 h, to maximize RNA yield

Centrifuge at 4 °C for 20 min at 12000 g

Remove the supernatant carefully

Wash with 1 mL of 70% Ethanol, flick the tube to release the pellet from the bottom of the tube

Centrifuge at 4 °C for 5 min at 7500 g

Discard the supernatant and air dry the RNA pellet

Critical-

After isopropanol precipitation, macrophage RNA needs to be incubated at − 80 °C for a minimum of 4 h, but the optimum time is 16 h

Avoid to dry the RNA completely to facilitate resuspension

Notes-

RNA can optionally be resuspended at 55 °C for 5 min before quantification

RNase inhibitors (e.g., Ribolock) can be added at a 1:20 dilution to prevent RNA degradation

Quantify RNA concentration with a fluorescent dye-based method such as the Qubit RNA HS Assay Kit. Briefly, dilute 1 μL resuspended RNA in a solution containing RNA HS reagent in a 1:200 dilution with Qubit RNA HS Buffer, incubate at RT for 2 min and quantify the RNA in a Qubit fluorometer. Calculate the RNA concentration taking into account the previously made dilution.

Pause point: RNA can be kept at − 80 °C for 2 weeks before proceeding with library construction; if samples are kept any further, we recommend to check RNA quality on a electrophoretic chip such as RNA 6000 Pico Kit (Agilent), or to run an RNase-free 0.8% agarose gel to confirm RNA integrity, visualizing the gel on a UV trans illuminator such as GelDoc or similar imaging system. Total RNA should appear as two sharp rRNA bands (28S and 18S) in a 2:1 ratio, while degraded RNA should appear as a smear.

What is the purpose of extracting RNA?

Why do we need to extract RNA rather than DNA?