RNA InstaPure System, 100ml Technical Data Sheet

Size: px

Start display at page:

Download "RNA InstaPure System, 100ml Technical Data Sheet"

Lewis Fletcher
5 years ago
Views:

For in vitro research use only RNA InstaPure System, 100ml Technical Data Sheet References: KP-0100-01 Storage: Store at 4 C. Protect from long exposures (days) to light.

1 For in vitro research use only RNA InstaPure System, 100ml Technical Data Sheet References: KP Storage: Store at 4 C. Protect from long exposures (days) to light. Introduction The isolation of pure and undegraded RNA from a large number of samples in a short time is a prerequisite for many experiments in molecular biology. Chirgwin et al. have shown(1) that using guanidinium thiocyanate and LiCl centrifugation yields good RNA from many tissues. This method turned out to be unsatisfying with tissues or cells containing high amounts of RNAse. Recent progress i.e. inclusion of phenol and chloroform has made it possible to obtain excellent RNA preparations within 1-2 hours free of DNA in a single-step procedure. This method provides very high yield and purity of RNA preparations. The ease of the RNA InstaPure method combined with excellent recoveries from small quantities of tissue or cells makes our product especially suitable for gene expression studies. RNA preparations are essentially undegraded, free of DNA and proteins and contain the entire spectrum of RNA molecules, including small (4-5 S) RNAs. The RNA is ready for Northern blotting experiments, dot blot hybridization, and polya + selection for mrna isolation. It can be directly used for molecular cloning, in vitro translation and RNAse protection experiments. Certain cells contain a material of unknown nature in the final RNA fraction (2). This material is readily soluble in water, insoluble in alcohol, forming DNA-like fibers and has an absorbance similar to nucleic acids in UV light. This material, most likely polysaccharides, is readily solubilized by guanidinium thiocyanate, seems not to interfere with hybridizations, but leads to wrong RNA estimations in the final preparations. In fact even cells in different developmental stages have been shown to contain different amounts of this contaminating material. A simple Extension of the standard RNA InstaPure procedure eliminates these impurities.

2 Reagents supplied RNA InstaPure 1 bottle with 100 ml Preparation Ready to use Storage Refrigerate at 2-8 C. Do not freeze. Stability Stable for nine month, refer to expiration date. Reagents required, but not supplied Chloroform p.a. Isopropanol p.a. 70% Ethanol p.a. RNA InstaPure Method RNA isolation by the RNA InstaPure method includes the following steps: 1. Homogenization RNA InstaPure (2 ml/100 mg tissue or 10 7 cells) 2. RNA Extraction 1 vol. homogenate vol. Chloroform 3. RNA Precipitation 1 vol. isopropanol 4. RNA Wash 70% ethanol Homogenization a. Homogenize tissue samples with RNA InstaPure (2 ml per 100 mg tissue) with a few strokes in a glass-teflon homogenizer. b. To isolate RNA from cells grown in suspension, sediment cells and lyse them by the addition of 0.2 ml RNA InstaPure per 10 6 cells. Cells grown in monolayer are lysed directly in the culture dish by the addition of RNA InstaPure (1 ml per 3.5 cm petri dish). Solubilize RNA by passing the lysate a few times through the pipette. RNA Extraction Add 0.2 ml chloroform per 2 ml of homogenate, cover tightly the samples, shake vigorously for 15 seconds and let them stay on ice (or at 4 C) for 5 minutes. After addition of chloroform and centrifugation t for 15 minutes at g (4 C) the homogenate forms two phases: the lower yellow phenol-chloroform phase and the colorless upper aqueous

3 phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 50% of the initial volume of RNA InstaPure plus the volume of tissue used for homogenization. RNA Precipitation Transfer the aqueous phase to a fresh tube, add an equal volume of isopropanol, mix and store the samples for 15 minutes at 4 C. Centrifuge samples for 15 minutes at g (4 C). RNA precipitate (often invisible before centrifugation) forms a white-yellow pellet at the bottom of the tube. RNA Wash Remove the supernatant and wash the RNA pellet once with 70% ethanol by vortexing and subsequent centrifugation for 8 minutes at g (4 C). Repeat the washing step if necessary (phenol smell!). Use at least 0.8 ml of ethanol per µg RNA. At the end of the procedure briefly dry the pellet under vacuum for 10 minutes. It is important not to let the RNA pellet dry completely, as it will greatly decrease its solubility. Dissolve the RNA pellet in 0.5% SDS or in 1 mm EDTA, ph 7 solution by vortexing or by passing it a few times through a pipette tip. An incubation for minutes at 60 C may be required to dissolve preparations of RNA. Diethylpyrocarbonate (DEPC)- treated RNAse free solutions (3) should be used for RNA solubilization. Notes and Comments a. Isolation of RNA from a small amount of tissue (1-10 mg). Homogenize samples in 0.8 ml RNA InstaPure, transfer the homogenates to Eppendorf tubes, add 80 µl of chloroform and store samples for 5 minutes at 4 C. Centrifuge samples in an Eppendorf centrifuge for 15 minutes, collect the aqueous phase and precipitate RNA with 0.4 ml of isopropanol for 45 minutes or overnight at 4 C. Centrifuge RNA precipitates for 15 minutes and wash once with 0.8 ml of 70% ethanol. b. Following isopropanol addition store samples overnight at 4 C in case the procedure has to be interrupted at this step. c. An additional precipitation might be necessary to use RNA isolated by the RNA InstaPure method in enzymatic assays. Following solubilization precipitate RNA in the presence of 0.2 M NaCl with one volume of isopropanol or with two volumes of ethanol for 1 hour at -20 C. d. Hands and dust may be the major source of RNAse contamination. Use gloves and keep tubes closed. The use of sterile disposable polypropylene tubes is recommended throughout the procedure.

4 e. Some commercial SDS preparations have acid ph. Adjust SDS preparation to ph if necessary. Trouble shooting Guide RNA isolation Expected yields of RNA per mg of tissue or 10 6 cells Liver and spleen 6-10 µg Kidney 3-4 µg Skeletal muscles and brain µg Placenta 1-4 µg Epithelial cells 8-15 µg Fibroblasts 5-7 µg Low yield Incomplete homogenization or incomplete lysis Final RNA pellet not completely dissolved A 260 /A 280 ratio < 1.65 Sample homogenized in too small volume of After homogenization samples were not stored at room temperature for 5 minutes The aqueous phase was contaminated with the phenol phase Incomplete dissolution of the final RNA pellet RNA degradation Tissues were not immidiately processed or frozen Cells were dispersed by trypsinization RNAse contamination was introduced during preparation Samples used for isolation were frozen at -20 C instead of -70 C Formaldehyd solution used for electrophoresis had a ph below 3.5 DNA contamination Samples homogenized in too small volume of Samples used for the isolation contained organic solvents (ethanol, DMSO, strong buffers or alkaline ph) Special Handling Precautions RNAPure contains an irritant (guanidinium thiocyanate) and poison (phenol). Handle RNAPure work with gloves. Do not get in eyes, skin or clothing. Avoid breathing vapor. In case of contact: Immediately flush eyes or skin with a large amount of water for at least 15 minutes and seek immediate medical attention.

5 Follow local or national waste disposal procedures. References 1. J.M. Chirgwin, A.E. Przybyla, R.J. MacDonald and W.J. Rutter, Biochemistry, 18, (1979) 2. E.I. Devinoy, C. Hubert, G. Jolivet, D. Thepot, N. Clergue, M. Desaleux, M. Dion, J.L. Serveley and L.M. Houdebine, Reprod. Nutr. Develop., 28, (1989) 3. T. Maniatis, E.F. Fritsch and J. Sambrock, Molecular Cloning, Cold Spring Harbor Laboratory, (1982)

TriFast FL Isolation of RNA, DNA and Protein simultaneously from Liquid Samples (FL)

TriFast FL Isolation of RNA, DNA and Protein simultaneously from Liquid Samples (FL) Cat.No. 30-2110 100ml 30-2120 200ml 30-2130 500ml Storage: Store at 4 C. Protect from long exposures (days) to light.