UPzol RNA Isolation Solution

Transcription

1 Product Insert UPzol RNA Isolation Solution LOT: See product label EXPIRY DATE: See product label ORDERING INFORMATION CAT. NO. SIZE PACKAGE CONTENT BR ml 200 ml UPzol RNA Isolation Solution COMPONENT UPzol RNA Isolation Solution COMPOSITION Single solution formulation for fast and easy RNA extraction. STORAGE 2 8 C or C in darkness (until expiry date see product label) FEATURES Simple and efficient RNA isolation from different sources in scalable volumes High-quality RNA preparation APPLICATIONS Isolation of RNA using a modified guanidine isothiocyanate/phenol method SPECIFICATIONS STARTING MATERIAL EXTRACTION TIME YIELD Tissue (100 mg), monolayer cells (3 cm dish), suspension of animal, plant, yeast or bacterial cells ( ) Approximately 1 h Depends on the type and amount of starting material PIN page 1

2 UPzol RNA Isolation Solution DESCRIPTION biotechrabbit UPzol RNA Isolation Solution is designed for efficient isolation of total RNA from animal tissues and cells, bacterial cells, plants and other material in variable amounts. The extraction method is based on a timesaving, one-step liquid phase separation. The UPzol RNA Isolation Solution is a mono-phase solution containing phenol and guanidine thiocyanate. After the addition of chloroform and subsequent centrifugation, the homogenate is separated into three phases: Colorless aqueous phase containing RNA (upper) White interphase (middle) Colored organic phase (lower) RNA is precipitated from the upper aqueous phase using alcohol. The UPzol RNA Isolation Solution provides high quality and high-integrity RNA, which can be used for all downstream applications, including northern analysis, cdna synthesis, RT-PCR, dot-blot hybridization, poly A+ selection, in vitro translation, cloning and RNase assays. MATERIALS SUPPLIED BY THE USER Chloroform (must be free of additives such as isoamyl alcohol) Isopropanol 75% ethanol (ice-cold) Polypropylene centrifuge tubes (for centrifugation at 12,000 g) RNase-free water, deionized formamide or 0.5% SDS for RNA solubilization and storage Optional: Glycogen IMPORTANT NOTE BEFORE STARTING To ensure high yields of good quality RNA, avoid repeated freezing and thawing of starting materials. page 2 info@biotechrabbit.com PIN

3 GUIDELINES FOR PREVENTION OF RNA DEGRADATION UPzol RNA Isolation Solution Special care should be taken to minimize contamination with RNases, as RNA is extremely sensitive to degradation. Always wear gloves, and change them frequently. Keep all tubes closed when possible. Keep samples and isolated RNA on ice. Reduce preparation time as much as possible. Use only sterile, disposable polypropylene tubes throughout the procedure (these tubes are generally RNase-free). Non-disposable plastic ware should be treated before use to ensure that it is RNase-free. Plastic ware should be thoroughly rinsed with 0.1 M NaOH, 1 mm EDTA followed by RNase-free water. You can also take chloroform-resistant plastic ware rinsed with chloroform to inactivate RNases. All glassware should be treated before use to ensure that it is RNase-free. o Glassware should be cleaned with detergent, thoroughly rinsed and oven baked at 240 C for four or more hours before use. Oven baking inactivates RNases and ensures that no other nucleic acids (such as plasmid DNA) are present on the surface of the glassware. o Autoclaving alone will not inactivate many RNases completely. The glassware should be immersed in 0.1% diethylpyrocarbonate (DEPC) solution for 12 h at 37 C before autoclaving or heating to 100 C for 15 min to remove residual DEPC. Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, rinsed with ethanol and finally allowed to dry. All buffers must be prepared with DEPC-treated RNase-free double-distilled water. Avoid handling bacterial cultures, cell cultures or other biological sources of RNases in the same lab where the RNA purification will be performed. Do not use equipment, glassware and plastic ware employed for other applications which might introduce RNase contaminations in the RNA isolation. SHORT PROTOCOL Applying UPzol to homogenize tissue samples, monolayer cells, or cell suspensions Add chloroform and incubate Phase separation Collect upper aqueous phase containing the RNA RNA precipitation, washing and re-solving PIN page 3

4 UPzol RNA Isolation Solution PROTOCOL FOR HOMOGENIZING TISSUE SAMPLES PROCEDURE Transfer up to 100 mg of tissue to a reaction tube. Add 1 ml UPzol RNA Isolation Solution. NOTES The sample volume should be < 10% of the UPzol RNA Isolation Solution volume. Homogenize the mixture. If possible, use a glass, Teflon or electrical homogenizer. Incubate the homogenized sample at room temperature for 5 min. Optionally, centrifuge at maximum speed at 4 C for 10 min to remove insoluble material. Transfer the supernatant to a new tube. The RNA remains in the supernatant, whereas insoluble debris and high-molecular-weight DNA will be pelleted. PROTOCOL FOR HOMOGENIZING MONOLAYER CELLS PROCEDURE Add 1 ml UPzol RNA Isolation Solution directly to a 3.5 cm cell culture petri dish. NOTES The volume of the UPzol RNA Isolation Solution depends on the size of the petri dish (1 ml per 10 cm²), not the number of cells in the dish. Mix by pipetting up and down repeatedly. Transfer complete mixture to a reaction tube. Incubate the homogenized sample at room temperature for 5 min. Optionally, centrifuge at maximum speed at 4 C for 10 min to remove insoluble material. Transfer the supernatant to a new tube. The RNA remains in the supernatant, whereas insoluble debris and high-molecular-weight DNA will be pelleted. PROTOCOL FOR HOMOGENIZING CELL SUSPENSIONS PROCEDURE Pellet up to cells by centrifugation. Remove the supernatant. Add 1 ml UPzol RNA Isolation Solution to the pellet and re-suspend. NOTES An additional washing step enhances RNA degradation and should be avoided. When using yeast or bacterial cells, an additional homogenization can be performed at this step. Incubate the homogenized sample at room temperature for 5 min. Optionally, centrifuge at maximum speed for 10 min at 4 C to remove insoluble material. The RNA remains in the supernatant, whereas insoluble debris and high-molecular-weight page 4 info@biotechrabbit.com PIN

5 Transfer the supernatant to a new tube. DNA will be pelleted. UPzol RNA Isolation Solution PROTOCOL FOR RNA ISOLATION PROCEDURE NOTES Add 0.2 ml chloroform for each milliliter UPzol RNA Isolation Solution added to the sample. Mix the sample by vortexing for 15 s. Incubate the sample at room temperature for 2 3 min. Centrifuge the sample at 12,000 g (10,000 rpm) for 15 min at 4 C to separate the phases. Transfer the upper aqueous phase carefully to a new centrifuge tube. The mixture separates into the following three phases: colorless aqueous containing RNA (upper, approx % of the total volume), white containing DNA and proteins (middle) and colored organic containing organic materials (lower). Avoid transferring inter phase material to avoid DNA contamination. Optionally, if very low (< 10 µg) RNA yield is expected, add 10 µg glycogen as carrier for each milliliter UPzol RNA Isolation Solution added to the sample. Add 0.5 ml isopropanol for each milliliter UPzol RNA Isolation Solution. Incubate the sample at room temperature for 10 min. Centrifuge at 12,000 g (10,000 rpm) for 10 min at 4 C. Remove the supernatant carefully and as completely as possible. Wash the pellet with1 ml ice-cold 75% ethanol. Centrifuge the sample at maximum speed for 10 min. Remove ethanol completely with a pipette. Incubate the tube with the lid open at room temperature for 2 3 min to allow the RNA pellet to dry. Dissolve the RNA pellet in water, deionized formamide or 0.5% SDS. Optionally, incubation at C for min improves solubilization. The RNA precipitate appears as a gelatinous pellet at the bottom of the tube. After washing the RNA pellet can be stored in fresh 75% ethanol for 1 3 weeks at 4 C or for 1 year at 20 C. Do not over dry the RNA pellet. Do not dry by vacuum centrifugation. Ensure the liquid is RNase-free before use. Do not use 0.5% SDS to solve the RNA if downstream assays include enzymatic reactions. Purified RNA can be used immediately. Store the RNA at 4 C (short-term) or 80 C (long-term). PIN page 5

6 UPzol RNA Isolation Solution TROUBLESHOOTING PROBLEM SOLUTION LOW YIELD Insufficient lysis or homogenization Insufficient dissolution of the RNA pellet Homogenize or lyse the starting material completely. Solve the RNA pellet completely. LOW A260/A280 RATIO Insufficient homogenization volume Contamination with phenol phase Insufficient dissolution of the RNA pellet Homogenize the sample in a bigger volume and incubate the sample at room temperature. Transfer the aqueous phase carefully. Solve the RNA pellet completely. DEGRADED RNA RNA source inappropriately handled or stored RNase contamination of solutions, tubes, etc. Ensure that the starting material is fresh. Ensure that the protocol especially the first steps has been performed quickly. Use sterile, RNase-free filter tips. Before every RNA preparation, clean the pipette, devices and working place. Always wear gloves. DNA CONTAMINATION Insufficient homogenization volume Homogenize the sample in a bigger volume. Samples may not contain organic solutions, e.g. ethanol, DMSO, concentrated buffers or an alkaline ph value. page 6 info@biotechrabbit.com PIN

7 SAFETY PRECAUTIONS UPzol RNA Isolation Solution UPzol RNA Isolation Solution contains phenol and guanidine isothiocyanate, which are harmful to health. Don t eat or drink components of the kit! The reagent should be handled by educated personal under an exhaust hood only! Always wear gloves while handling this reagent. Avoid skin contact! In case of contact, flush eyes or skin with a large amount of water for at least 15 min. Seek medical care immediately! Handle and discard waste according to local safety regulations! Do not add bleach or acidic components to the waste after sample preparation! Contact with acids liberates very toxic gas! PIN page 7

8 UPzol RNA Isolation Solution CERTIFICATE OF ANALYSIS Functionally tested for RNA quality by agarose gel electrophoresis and RT-PCR. Quality confirmed by: Head of Quality Control SAFETY INSTRUCTIONS For safety instructions please see Safety Data Sheets (SDS)/Sicherheitshinweise finden Sie in den SDS unter: USEFUL HINTS Visit Applications at for more products and product selection guides. Most biotechrabbit products are available in custom formulations and bulk amounts. CONTACT BIOTECHRABBIT biotechrabbit GmbH Volmerstr. 9a Berlin, Germany Phone: Fax: Legal Disclaimer and Product Use Limitation Purchase of product does not include a license to perform any patented applications; therefore it is the sole responsibility of users to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used. This product was developed, manufactured, and sold for in vitro use only. It is not suitable for administration to humans or animals. Trademarks: biotechrabbit, UPzol (biotechrabbit GmbH). valid from page 8 info@biotechrabbit.com PIN