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MOST COMMONLY ASKED QUESTIONS ABOUT RNA EXTRACTION REAGENTS

WHAT HAPPENED TO RNAzol B????????????????????

The RNAzol B has been upgraded and its name has been changed to RNA-BEE.

The RNA-Bee is the same as RNAzol B - the only difference is that the procedure time has been cut from 1-½ hours to 1 hour. It still is the same color.

Effective August 1, 2001, the RNAzol B will no longer be available.

WHY DID WE NOT RECEIVE ANY NOTICE???

The RNAzol B was a patient product and the ownership of the name was in question. Unfortunately, the doctor that owns the product was ordered to discontinue the usage of the RNAzol B trademark; therefore, from a legal point we can no longer use the trade name, which was effective immediately.

HAS THE PROCEDURE CHANGED???

I can fax you an RNA-Bee protocol immediately for your review and if you have any questions please call back for technical assistance.

There are free 15 ml samples available. The price for FedEx is $40.00 for 2nd day delivery.

MOST COMMONLY ASKED QUESTIONS
ABOUT RNA EXTRACTION REAGENTS

1. I have a low 260/280 ratio; why is this occurring?
2. When I run my electrophoretic argarose gel I see DNA contaminations; why is this occurring?
3. Why is my aqueous phase colored?
4. Can I use extremely small sample volumes?
5. What is the total yield amount of RNA expected?
6. How many cells or how much tissue can I use with RNAzol?
7. What is the expected yield for mRNA (messenger RNA)?
8. What is the optimum storage conditions for my sample?
9. I am in the middle of performing this RNA extraction analysis and I can't finish the procedure. Where is a good point so that I can salvage my experiment?
10. What are the basic components of RNAzol , RNAzol B, or the STAT-60? Or in order to properly dispose of my waste solutions of RNAzol, my Safety Officer needs to know the components or chemical makeup.
11. What kinds of sample tubes can I use for my RNA extraction's that is compatible with phenol?
12. What is the advantage of using LS-50 for liquid samples over the other extraction reagents (RNAzol, RNAzol B, and STAT-60)?
13. What is the basic difference between RNAzol, RNAzol B,   and STAT-60?


RNAzol Technical Questions
I. Low 260/280 ratio: A. A overly dried pellet. 1. Decant ethanol wash after centrifugation and AVOID distributing the pellet. 2. Position the tube upright and air dry at room temperature for 10-15 minutes. This time limit should prevent ethanol contamination. 3. It is necessary to blot tube after decanting step using only sterilized paper. DO NOT USE A SPEED VAC -- it will have the tendency to overly dry the pellet which will also make the pellet insoluble. B. Contamination with organic layer during aqueous phase removal. 1. It may be necessary to sacrifice QUANTITY to increase/improve the QUALITY of RNA. 2. Depending on the volume of the aqueous phase, leave about 50-100ul on the organic layer to prevent intermixing. 3. Aspirating the aqueous phase too fast or withdrawing the pipette tip too fast may cause contamination even though visually it may seem like there has been no contact with the organic layer. C. Sample may contain many polysaccharides, glycogen, fats, or lipids. 1. It is recommended to wash the sample with a solution of 4 Molar LiCl (lithium chloride) prior to the ethanol wash. D. The limit of acceptability for the 260/280 ratio is >1.6<2.1.1 If lower than 1.6 - you may want to consider a re-extraction with RNAzol, RNAzol B or STAT-60 depending on which one is being used. 2. The re-extraction step consists of the addition of RNAzol reagent to the aqueous phase after the initial extraction with chloroform. 3. After aqueous phase has been removed from the initial extraction step to a separate eppendorf tube or suitable vial, add an equal volume of the RNAzol extraction reagent and continue with the appropriate amount of chloroform (the same amount added in the primary step). 4. Extending the homogenization step from 5-15 minutes may be helpful in increasing low 260/280 ratio. 5. NEVER use a vortexer in the homogenization or chloroform extraction step-using one will decrease the 260/280 ratio. 6. IF greater than 2.1 - your RNA has been degraded, probably due to prolonged exposure to the extraction reagent. SEE IIIV. B, 1-3.
NOTE: *The 260/280 is a way in which one can measure the purity or quality of the RNA extracted. *The absorbance from a spectrophotometer at 260 nm (wavelength) is obtained and then divided by the absorbance obtained at 280 nm to determine a ratio. It should be between 1.6 to 2.0.

II. Causes of DNA contamination: A. Getting too close to DNA organic layer during aqueous phase removal. B. Vortex -- Do not vortex during homogenization. Vortex is OK during Isopropanol addition. C. Homogenization step is not long enough. Try extending the homogenization step to 5-10 minutes, but not exceeding 25 minutes.

III. Colored aqueous phase: A. Reason that aqueous phase is colored (red, blue, or pink) is because the sample contains a high amount of adipose (fat) tissue, polysaccharides, or lipids. 1. If this colorization of the aqueous phase occurs, it indicates you still have phenol trapped in this aqueous layer. Decrease the amount of RNA extraction reagent and increase the amount of chloroform. 2. Normally if this occurs, your experiment is lost. To date there is no known method to isolate quality RNA from adipose tissue. Call for more details.

IV. Small sample volumes: A. When using small sample volumes (1,000 - 10,000 cells), you may want to purchase a glycogen carrier solution from Boehringer. Call Boehringer for their protocol and procedure. Phone number is 1-800-428-5074.

V. Amount of RNA/DNA expected: A. Per 100 mg of tissue or 10 million cells, you should recover between 100-150 ug of total RNA. B. Using liver you can expect 5 times as much up to 750ug. You may want to increase the amount of RNA extraction reagent in your homogenization step.

VI. Amount of tissue or cells that can be used: A. There is no limit if the ratio of 2 ml of RNAzol or RNAzol B per 100 mg of tissue or 10 million cells adhered to. The ratio must remain the same. Or 1 ml of STAT-60 per 100 mg of tissue or 10 million cells.

VII. Expected yield of mRNA (messenger): A. The expected yield of mRNA is always 2 - 3% of total RNA because that's all the mRNA there is in total RNA. B. Per 100ul expect 2 - 3ul of mRNA.

VIII. Storage conditions: A. Reagents-RNAzol, RNAzol B, STAT-60, LS 50, and DNA STAT-60. 1. These reagents need to be stored in refrigerated temperatures away from direct light (long-term). Occasional exposure to light is of no concern. 2. RNAzol upon long exposure to light will develop a deep yellow/light tan color which indicates the breakdown of phenol in the RNAzol solution. 3. Under the appropriate storage conditions, RNAzol, RNAzol B, and STAT-60 have been noted to go 1 - 2 years beyond the expiration date printed on the bottom. B. storage temperature for samples is -70 C freezer. 3. Store harvested tissue in -70 C as soon as harvested to reduce Rnase contamination. Contamination is still likely if RNAzol B or STAT-60 are not stored with the samples.

IX. Interruption points in RNA/DNA extractions: A. The best place to stop in the RNA extraction procedure is during the homogenization step and placed in -70 C freezer (optimum storage conditions). B. During the precipitation step (Isopropanol addition), one can store Isopropanol plus sample into -70 C freezer, not exceeding 48 hours.

X. The basic components of RNAzol, RNAzol B, STAT-60: Due to the fact the RNA extraction reagents are patented this information is considered proprietary and is not to be given out. You can only give out information that has been published. 1. <50% concentration of phenol2. 2. Molar concentration of GTC (Guanidine thiocyanate).

XI. Different kinds of sample tubes that are compatible with RNAzol: A. Eppendorf 1.5-2.0ml conical spin tubes are the best to use. B. For larger volumes El Kay manufactures a high density polypropylene tube for 10-50ml or more call El Kay for more information. Phone number is 1-800-522-7763.

XII. The advantage of using LS-50 for liquid samples over the other RNA extractions: A. LS-50 is exclusively designed for the extraction of RNA from liquid samples i.e., whole blood, CSF (Cerebral Spinal Fluid), urine, Plural fluid, or Synovial fluid. Using LS-50 requires a 1:4 dilution of sample to LS-50: for example 1ml of whole blood plus 3ml of LS-50. B. For RNAzol or RNAzol B, you have to use 1:10 to 1:15 dilution (1ml of sample plus 9ml of RNAzol or RNAzol B). C. For STAT-60 you have to use a 1:15 - 1:20 dilution (1ml of sample plus 14ml of STAT-60).

XIII. The basic difference between RNAzol, RNAzol B, and STAT-60: A. RNAzol is our first generation extraction reagent developed using the phenol/chloroform method for the extraction of total RNA or mRNA. 1. The procedure basically takes about 3 hrs. to perform. B. RNAzol B is our 2nd generation extraction reagent for total RNA and mRNA. 1. RNAzol B has a blue indicator that helps you visualize the difference during the Chloroform extraction step between the clear aqueous layer and the blue organic layer. 2. RNAzol B takes only 1.5 hrs. to perform as compared to 3 hrs. using RNAzol. C. STAT-60 is our 3rd generation extraction reagent. 1. STAT-60 has a red indicator to help visualize the aqueous and organic layer. 2. STAT-60 takes only 60 minutes to perform as compared with 3 hrs using RNAzol and 1.5 hrs. using RNAzol B.

NOTE: *The quality and quantity of RNA extracted from RNAzol and RNA-Bee is identical. *The basic principle of RNA/DNA extraction is based on pH. *At an acid pH, RNA is soluble in the aqueous phase. The DNA and Proteins are insoluble and are located in the organic layer. *At an alkaline pH, DNA is soluble in the aqueous phase while RNA is insoluble.