Talk:Preparation of RNA Samples from Mouse Tissues

Original Protocol from Invitrogen Website

Preparing Wash Buffer II with Ethanol

Before beginning lysis, add 60 ml 96-100% ethanol to Wash Buffer II. Check the box on the Wash Buffer II label to indicate that ethanol was added. Store Wash Buffer II with ethanol at room temperature.

Lysate Preparation with TRIzol® Reagent

Use TRIzol® Reagent to prepare lysates from various sample types as described below. Tissues Homogenize tissue samples in 1 ml TRIzol® Reagent per 50–100 mg tissue using a tissue homogenizer or rotor-stator. The sample volume should not exceed 10% of the volume of TRIzol® Reagent used for homogenization.

Adherent Cells

Lyse cells directly in a culture dish by adding 1 ml of TRIzol® Reagent to the dish and passing the cell lysate several times through a pipet tip. The amount of TRIzol® Reagent required is based on the culture dish area (1 m per 10 cm2) and not on the number of cells present.

Suspension Cells

Harvest cells and pellet cells by centrifugation. Use 1 ml of the TRIzol® Reagent per 5–10 × 106 animal, plant, or yeast cells, or per 1 × 107 bacterial cells. Lyse cells by repetitive pipetting up and down. Do not wash cells before addition of TRIzol® Reagent to avoid any mRNA degradation. Disruption of some yeast and bacterial cells may require a homogenizer.

Phase Separation

Following cell or tissue lysis (above), perform the following steps to isolate the RNA.

Incubate the lysate with TRIzol® Reagent (above) at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes.

Add 0.2 ml chloroform or 50 μl 4–Bromoanisole per 1 ml TRIzol® Reagent used. Shake the tube vigorously by hand for 15 seconds. Note: Vortexing may increase DNA contamination of your RNA sample. Avoid vortexing if your downstream application is sensitive to the presence of DNA or perform a DNase-digestion step during RNA purification or after purification Refer to the PureLink™ RNA Mini Kit manual, available from our web site at www.invitrogen.com.

Incubate at room temperature for 2–3 minutes.

Centrifuge the sample at 12,000 × g for 15 minutes at 4°C. Note: After centrifugation, the mixture separates int a lower, red phenol–chloroform phase, an interphase, and a colorless upper aqueous phase which contains the RNA. The volume of the aqueous upper phase is ~600 μl.

Transfer ~400 μl of the colorless, upper phase containing the RNA to a fresh RNase–free tube.

Add an equal volume of 70% ethanol to obtain a final ethanol concentration of 35%. Mix well by vortexing.

Invert the tube to disperse any visible precipitate that may form after adding ethanol.

Proceed to Binding, Washing and Elution, below.

Binding, Washing and Elution

Follow the steps below to bind, wash, and elute RNA from your sample.

Transfer up to 700 μl of sample (prepared as described above) to a Spin Cartridge (with a Collection Tube).

Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow–through and reinsert the Spin Cartridge into the same Collection Tube.

Repeat Steps 1–2 until the entire sample has been processed. Optional: If your downstream application requires DNA-free total RNA, proceed to On–Column PureLink™ DNase Treatment During RNA Purification at this time (see the PureLink™ RNA Mini Kit manual, available from our web site at www.invitrogen.com, for details).

Add 700 μl Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through and the Collection Tube. Insert the Spin Cartridge into a new Collection Tube.

Add 500 μl Wash Buffer II with ethanol (previous page) to the Spin Cartridge.

Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow–through, and reinsert the Spin Cartridge into the same Collection Tube.

Repeat Steps 5–6 once.

Centrifuge the Spin Cartridge and Collection Tube at 12,000 × g for 1 minute at room temperature to dry the membrane with attached RNA. Discard the Collection Tube and insert the Spin Cartridge into a Recovery Tube.

Add 30 μl–3 × 100 μl (3 sequential elutions with 100 μl each) RNase–Free Water to the center of the Spin Cartridge (refer to the PureLink™ RNA Mini Kit manual for more details, available from our web site at www.invitrogen.com).

Incubate at room temperature for 1 minute.

Centrifuge the Spin Cartridge with the Recovery Tube for 2 minutes at ≥12,000 × g at room temperature. Note: If you are performing sequential elutions, collect all elutes into the same tube.

Storage and Downstream Applications of Purified RNA

Store your purified RNA on ice if you will use the RNA within a few hours. For long–term storage, store your purified RNA at –80°C.