Bio-Rad RNA Fatty and Fibrous Tissue Kit User Manual

Aurum

™

Total RNA Fatty and

Fibrous Tissue Kit

Instruction Manual

Catalog # 732-6830

The Aurum Total RNA Fatty and Fibrous Tissue Kit is composed of:

Module 1 – 732-6870 (Aurum Total RNA Fatty and Fibrous Tissue Module) Module 2 – 732-6880 (PureZOL

packaged and shipped separately

™

RNA Isolation Reagent, 50 ml),

For technical support, call your local Bio-Rad office, or in the US, call 1-800-4BIORAD (1-800-424-6723).

Table of Contents

Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Section 2 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .2

Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .3

Section 4 Materials and Equipment Required

(Not Provided in the Kit) . . . . . . . . . . . . . . . . . . .3

Supplies for Tissue Grinding, Disruption,

and Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Additional Equipment Required for Vacuum Format . . . .4

Section 5 Before Using the Aurum™

Section 6 Vacuum Manifold Setup and Use With the

Total RNA Fatty and Fibrous Tissue Kit . . . . . . .5

Maximum Starting Material Amounts . . . . . . . . . . . . . . .5

Minimum Starting Material Amounts . . . . . . . . . . . . . . . .5

Reagents Used With the Aurum Total RNA

Fatty and Fibrous Tissue Kit . . . . . . . . . . . . . . . . . . . . . .7

Maintaining an RNase-Free Environment . . . . . . . . . . . .8

Sample Disruption and Homogenization . . . . . . . . . . . .9

Column Adaptor Plate . . . . . . . . . . . . . . . . . . .11

Guidelines for Vacuum Format . . . . . . . . . . . . . . . . . . .11

About the Column Adapter Plate (CAP) . . . . . . . . . . . .11

Preparing the Aurum Vacuum Manifold . . . . . . . . . . . .11

Vacuum Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Manifold Wash Setup . . . . . . . . . . . . . . . . . . . . . . . . . .13

Section 7 Vacuum Protocol . . . . . . . . . . . . . . . . . . . . . . .14

Section 8 Spin Protocol . . . . . . . . . . . . . . . . . . . . . . . . . .20

Section 9 Troubleshooting Guide . . . . . . . . . . . . . . . . . . .25

Section 10 Ordering Information . . . . . . . . . . . . . . . . . . . .32

Section 1 Introduction

The Aurum™ total RNA fatty and fibrous tissue kit produces high yields of pure total RNA from samples that are difficult to disrupt. The kit is ideal for fatty or fibrous tissues, or samples that are rich in RNases. It also works well with most animal and plant tissues, cultured cells, yeast, and gram-negative and gram-positive bacteria. With this kit, greater than 100 µg of total RNA can be isolated from many sample types. Total RNA samples isolated using the Aurum total RNA fatty and fibrous tissue kit are suitable for use in a variety of downstream applications, including reverse transcription-PCR (RT-PCR), realtime PCR, in vitro translation, northern blots, and microarray analysis.

The Aurum total RNA fatty and fibrous kit uses a quick, easy-to-follow procedure for the purification of total RNA from difficult-to-disrupt samples. First, samples are disrupted and lysed using the PureZOL™ RNA isolation reagent. Subsequently, chloroform is added to the lysate, mixed, and centrifuged to achieve separation of the organic and aqueous phases. The aqueous phase, which contains the RNA, is carefully recovered and mixed with ethanol. The sample is then passed through a silica membrane packed in the Aurum RNA binding mini column, where nucleic acids get bound. Wash steps are performed to remove proteins and other cellular debris. An optional on-column DNase I digest is performed to remove any remaining genomic DNA. The RNA, which is eluted with the elution solution supplied in the kit, is now ready for downstream applications without further manipulation.

The membrane in the Aurum RNA binding mini column selectively binds to mRNA and larger rRNAs, while small RNA molecules less than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, and tRNA (which together comprise 15–20% of total RNA), are removed. The on-column DNase l digest and subsequent wash steps that are performed during the purification effectively remove genomic DNA contamination as well as the residual DNase enzyme, eliminating the need for separate and lengthy DNase treatments and DNase removal protocols on the eluted RNA.

The Aurum total RNA fatty and fibrous tissue kit is designed to isolate total RNA from various amounts of tissue (5–100 mg) and cells (50–2.4 x 10 cells)*. The kit includes sufficient reagents and columns for 50 purifications. All solutions and RNA binding mini columns in the kit are RNase-free, ensuring the integrity of the isolated RNA. The Aurum total RNA fatty and fibrous tissue kit may be used in a spin format, or in a vacuum format using the Aurum vacuum manifold (catalog # 732-6470).

* The amount of starting material may be less for certain types of samples. For more detail, refer to

Section 5.

Section 2 Kit Components

The Aurum™ total RNA fatty and fibrous tissue kit contains the following components:

Components* Quantity/Amount

RNA binding mini columns 50 Capless wash tubes, 2.0 ml 50 Capped microcentrifuge tubes, 1.5 ml 50 Capped microcentrifuge tubes, 2.0 ml 100 DNase I (lyophilized) 1 vial Low stringency wash solution 20 ml (5x concentrate) High stringency wash solution 40 ml Elution solution 20 ml DNase dilution solution 20 ml PureZOL™ RNA isolation reagent** 50 ml (packaged and shipped separately)

*There may be reagent remaining in the some bottles. **PureZOL RNA isolation reagent contains poison (phenol) and an irritant (guanidine thiocyanate). The

reagent causes burns and can be fatal if ingested. When working with PureZOL, use gloves and eye protection (lab glasses, shield, and safety goggles). Do not get on skin or clothing. Avoid breathing vapor. Read warning notice on bottle and MSDS.

Section 3 Storage Conditions

Store components of the kit at the recommended temperatures (see Table 1 below).

Table 1. Recommended storage temperature for the Aurum™ total RNA fatty and fibrous tissue kit components.

Kit Components Storage Temperature

PureZOL™ RNA isolation reagent Store at 4–25°C

Low stringency wash buffer Room temperature (before and after the

addition of ethanol)

High stringency wash buffer Room temperature

Elution solution Room temperature

DNase dilution solution 4°C; allow solution to equilibrate to

room temperature before use

DNase I (lyophilized) Room temperature in lyophilized form;

once reconstituted in 250 µl of 10 mM Tris, pH 7.5, aliquot and store at –20°C in a nonfrost-free freezer. Do not freezethaw more than once after reconstitution

Aurum RNA binding mini columns Room temperature and microcentrifuge tubes

Section 4 Materials and Equipment Required (Not Provided in the Kit)

• Microcentrifuge, capable of spinning at >12,000 x g at 4°C and room temperature

• 95–100% ethanol, ACS grade or better

• Tris for DNase I reconstitution (catalog #161-0716)

• Chloroform (without additives such as isoamyl alcohol) for phase separation; 0.2 ml of chloroform per 1 ml of PureZOL required

Supplies for Tissue Grinding, Disruption, and Homogenization

• Fresh tissue: tissue cutter

• Frozen tissue: liquid nitrogen, mortar and pestle

• Tissue homogenizer: rotor-stator homogenizers, or bead mill homogenizers recommended

Additional Equipment Required for Vacuum Format

• Aurum vacuum manifold with vacuum regulator and column adaptor plate (catalog # 732-6470), or other vacuum manifold with luer fittings.

• Vacuum source (capability of –23 inHg required)

Section 5 Before Using the Aurum™ Total RNA Fatty and Fibrous Tissue Kit

Please read the following guidelines before proceeding with the total RNA isolation.

Maximum Starting Material Amounts*

The Aurum total RNA fatty and fibrous tissue kit is designed to process up to the amounts indicated below (per column):

•1 x 10

• One 10

•2.4 x 10

•3.0 x 10

• 100 mg of animal tissue (a 4 mm cube of most animal tissue weighs

• 100 mg of plant tissue

• 50 mg filamentous fungi

Warning: Processing larger amounts of starting material may lead to column clogging and reduced RNA purity. It is crucial that the appropriate amount of starting material be used. For samples that are known to be rich in RNA, it is highly recommended that less than the maximum amount of starting material be used so that the binding capacity of the column is not exceeded. In addition, complete disruption and homogenization of the starting material is critical to prevent column clogging and reduced RNA yields.

cultured mammalian cells grown in suspension

cm plate mammalian cultured cells grown in monolayer

of gram-positive or gram-negative bacteria (equivalent to

3 OD•ml of bacteria)

of yeast (equivalent to 3 OD•ml of yeast)

approximately 70–85 mg)

Minimum Starting Material Amounts

• 50 cultured mammalian cells

• 5 mg of animal tissue

• 5 mg plant tissue

* Spectrophotometric determination of bacterial or yeast culture density is a REQUIREMENT for optimal total RNA isolation from these starting materials. To determine the density of a bacterial or yeast culture (OD 50 µl of culture with 950 µl growth medium (20-fold dilution). Use the growth medium as a blank and take the spectrophotometric reading at 600 nm. Multiply this figure by 20 to calculate the OD bacterial or yeast culture. Depending upon the OD of the culture will be selected to provide an optimum amount of bacteria or

value of the undiluted

600

value, a specific volume

600

), combine

600

yeast for processing. To calculate the volume of culture required, use the following equation:

(OD

of undiluted culture) x (culture volume in ml) = # OD•ml

600

For example, 3 OD•ml of yeast would require 500 µl of an undiluted culture with an OD

= 6.

600

Note: 1 OD

1 x 10

is equivalent to approximately 8 x 108bacterial cells/ml, or

600

yeast cells/ml.

Table 2. Yield (per column) of total RNA from various samples using the Aurum total RNA fatty and fibrous tissue kit.

Average Yield

Starting Material (Amount Used)* (µg)**

Cultured cells (1 x 10

)

293H 145

Bacteria (2.4 x 10

or 3 OD•ml)

E. coli 30

Yeast (3 x 10

or 3 OD•ml)

S. cerevisiae 64

Fatty animal tissue (100 mg)

Brain 96 Breast 58 Adipose 14

Fibrous animal tissue (100 mg)

Heart 85 Cartilage 54 Skin 63

Plant tissue (100 mg)

Potato 91 Arabidopsis 5

Filamentous fungi (50 mg)

Aspergillus niger 12

* Starting material amounts in parentheses are the maximum amounts recommended for this kit. Note: The

elution volume should be decreased to 30 µl if only a small amount of starting material (<500,000 cells or <10 mg of animal or plant tissue) is used.

** Yield figures are representative of a minimum of 20 mini column preps performed in both vacuum and spin

formats.

Reagents Used With the Aurum Total RNA Fatty and Fibrous Tissue Kit

PureZOL™ RNA Isolation Reagent for Sample Lysis

Use 1 ml of PureZOL for up to:

• 100 mg of tissue

•1 x 107cultured cells grown in suspension

• One 10 cm2plate of cultured cells grown in monolayer

•2.4 x 109of gram-positive or gram-negative bacteria (equivalent to 3 OD•ml of bacteria)

•3 x 10

Low Stringency Wash Solution

• The low stringency wash solution is provided as a 5x concentrate. Add 4

DNase I

• 10 mM Tris, pH 7.5 prepared in DEPC-treated water (not supplied) is

• Reconstitute the lyophilized DNase I by adding 250 µl of 10 mM Tris, pH

of yeast (equivalent to 3 OD•ml of yeast)

volumes (80 ml) of 95–100% ethanol to the low stringency wash solution concentrate before initial use

required to reconstitute the RNase-free DNase I that is provided as a lyophilized powder

7.5 to the vial and mix by briefly pipetting up and down. Do not vortex

• Aliquot and store the reconstituted DNase I at –20°C in a nonfrost-free freezer. Avoid freeze-thaw cycles

Note: 5 µl of the DNase I stock is needed per column or prep. When the DNase is ready to be used, it must be mixed with 75 µl of the DNase dilution solution (provided in the kit) per column. Once diluted with DNase dilution solution, use the DNase immediately and do not refreeze for later use

Elution Guidelines

• Apply elution solution directly to the membrane stack at the base of each RNA binding mini column

Preparation of DEPC-Treated Water

• Autoclaving of laboratory solutions and buffers used for RNA preparation does not guarantee the complete inactivation of RNases, which can maintain residual activity causing RNA samples to be degraded. For this reason, solutions and buffers should be treated with diethyl pyrocarbonate

(DEPC) to inactivate RNases. DEPC is an efficient, strong, and nonspecific RNase inhibitor that is usually used at a concentration of 0.1%

• To prepare a 0.1% (v/v) solution of DEPC-treated water, add 1.0 ml of liquid DEPC per 1 L of water. Incubate the solution at 37°C for 1 hr while mixing thoroughly. Autoclave the treated water to remove the DEPC

• Warning: DEPC is suspected to be a carcinogen and should be handled with care. Always use gloves and open under a fume hood

Preparation of 10 mM Tris, pH 7.5, for DNase I Reconstitution

• To prepare 50 ml of 10 mM Tris, pH 7.5, add 60.6 mg of Tris (catalog # 161-0716) to 45 ml of DEPC-treated water. Mix until Tris is completely dissolved. Adjust the pH of the solution by dropwise addition of 6N HCl. Once the pH is adjusted to 7.5, add more DEPC-treated water to make a final volume of 50 ml

Note: DEPC is destroyed by primary amines (e.g., Tris). If a solution containing a primary amine will be DEPC-treated, omit the amine in preparing the solution. Perform the DEPC treatment as described above and add the amine to the autoclaved solution once the solution has cooled

Preparation of 70% Ethanol

• To prepare 100 ml of 70% ethanol, add 70 ml of 95–100% ethanol to 30 ml of DEPC-treated water. Mix well before use.

Maintaining an RNase-Free Environment

• Although the components of this kit are provided free of contaminating ribonucleases, great care must be taken not to contaminate the solutions or the RNA binding columns. Gloves should always be worn when handling RNA and should be changed frequently. Proceed through the RNA isolation as quickly as possible with care

• Solutions that are prepared by the user should be treated with DEPC to inactivate RNases as described above.

+ 28 hidden pages