body fluid RNA_product_prodcut insert
 
DNA/RNA Extraction Kits
 
Orochem offers a complete line of kits in individual spin columns and 96-well formats for DNA/RNA extraction from a variety of sample sources such as bacteria, plant, blood, serum, tissue and forensic samples.
 
From Bacteria (Plasmid, Genomic): High yield of Plasmid DNA in less than 20 minutes
 
From Blood: Available in a spin column or 96 well formats for blood or serum.
 
From Tissue: For all kinds of tissues including mouse tails. Fast protocol and methodology eliminates hands-on homogenization plus no hazardous organic solvents required.
 
From Plant: Completely removes all polysaccharide and phenolic inhibitors of PCR from plant material. Can be used with seeds, leaves, bark and roots.
 
Forensic Applications: DNA for Forensic Applications!
 
  Call for FREE SAMPLES!
 
 
 
GenQuikTM Body Fluid RNA Extraction Kit                                                                 Page | 2
 
 Tel.: 630-916-0225 Fax: 630-916-0250 www.orochem.com Orochem Technologies Inc 331 Eisenhower Ln South, Lombard, IL 60148
GenQuikTM Body Fluid RNA Extraction Kit For total RNA (Cerebrospinal fluid, plasma, saliva, serum, sputum, synovial fluid, urine, whole blood) Catalog # OGQRNABF
 
Introduction The GenQuikTM Body Fluid RNA Extraction Kit is intended to provide efficient purification of total RNA from cerebrospinal fluid, plasma, saliva, serum, sputum, synovial fluid, urine, whole blood. The RNA remains soluble in the supernatant and is captured on a membrane spin filter while contaminants are passed through the filter by centrifugation. The filter is washed to remove any remaining contaminants and salts. The RNA is then eluted into certified RNasefree water (provided). RNA purified by this method is of high quality and free from contaminants and PCR inhibitors, and is particularly suitable for demanding PCR/RT-PCR analyses, and microarray analysis.
 
Required Equipment: • Homogenizer • Microcentrifuge (12,000 x g) • Vortex • RNase-free pipette tips  • RNase-free tubes 
 
Preparation of RNA When preparing RNA, samples should be processed quickly during the manual steps of the procedure. Extreme care should be taken to avoid contamination with RNases when handling Buffers. Wear gloves.
 
Sample storage After collection and centrifugation, plasma or serum can be stored at 2–8°C for up to 6 hours. For long-term storage, freezing at –20°C or – 80°C in aliquots is recommended.
GenQuikTM Body Fluid RNA Extraction Kit                                                                 Page | 3
 
 Tel.: 630-916-0225 Fax: 630-916-0250 www.orochem.com Orochem Technologies Inc 331 Eisenhower Ln South, Lombard, IL 60148
Frozen plasma or serum samples must not be thawed more than once. Repeated freeze–thawing leads  to reduced yields of RNA. 
 
Kit Storage: Storage conditions for Kit reagents and components should be at room temperature (15-30°C) for 1 year.
 
Kit Contents:
 
Component  Description
 
Lysis Buffer (RBF1)
 Binding Buffer (RBF2)  Wash Buffer (RBFW)  Elution Buffer (RBFE) (RNase-free water) Spin Filters Spin filter basket in a 2.0 ml tube. 2-mercaptoethanol 1mL/vial Carrier RNA 600µg/vial (Lyophilized powder)
 
All buffers are completely stable for a period of at least 12 months from the date of receipt when stored under ambient conditions. Please avoid exposure to direct sunlight or extremes in temperature. 
 
Precaution: While working with buffers, always wear safety glasses, laboratory coat and gloves. Be careful to avoid contact with eyes and skin. In the case of such contact, wash immediately with water. 
 
Handling of Spin columns Because of the sensitivity of nucleic acid amplification technologies, the following precautions are necessary when handling spin columns in order to avoid cross-contamination between sample preparations:
 
• Carefully apply the sample or solution to the spin column. Pipette the sample into the spin column without wetting the rim of the column.  • Change pipette tips between all liquid transfers. The use of aerosol-barrier pipette tips is recommended. 
GenQuikTM Body Fluid RNA Extraction Kit                                                                 Page | 4
 
 Tel.: 630-916-0225 Fax: 630-916-0250 www.orochem.com Orochem Technologies Inc 331 Eisenhower Ln South, Lombard, IL 60148
• Avoid touching the spin membrane with the pipette tip.  • After all pulse-vortexing steps, briefly centrifuge the microcentrifuge tubes to remove drops from the inside of the lid.  • Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.
 
Preparation before experiment 1. Add amount of ethanol specified on the bottle label to the buffer RBFW concentrate before using this kit. Either 100% or 95% (denatured) ethanol can be used. Mix well and store at room temperature.  2. Prepare Buffer RBF1 fresh for each use by adding 2- mercaptoethanol (add 10 μl of 2-mercaptoethanol for 1 ml Buffer (RBF1). 3. Carrier RNA- Add 600ul of Rnase and Dnase free water to the tube containing 600ug of carrier RNA to obtain 1ug/ml, make aliquots and store at -20°C. Carrier RNA serves two purposes. Firstly, it enhances binding of nucleic acids to the membrane, especially if there are very few target molecules in the sample. Secondly, the addition of large amounts of carrier RNA reduces the chance of RNA degradation in the rare event that RNase molecules escape denaturation by the detergent in lysis Buffer.  4. Addition of internal controls internal control RNA should be added together with the carrier RNA to the lysis buffer. For optimal purification efficiency, internal control molecules should be longer than 200 nucleotides, as smaller molecules are not efficiently recovered.
 
Protocol 1. Place 0.5 ml of whole body fluid sample cerebrospinal fluid, plasma, saliva, serum, sputum, synovial fluid, urine, whole blood to a 1.5 ml RNase–free microcentrifuge tube. 2. Add 0.5 ml Lysis Buffer RBF1prepared with 2-mercaptoethanol (10ul of 2-mercaptoethanol/ml lysis buffer). Add 10 ul ( 1ug/ul) of carrier RNA solution.
GenQuikTM Body Fluid RNA Extraction Kit                                                                 Page | 5
 
 Tel.: 630-916-0225 Fax: 630-916-0250 www.orochem.com Orochem Technologies Inc 331 Eisenhower Ln South, Lombard, IL 60148
3. Vortex thoroughly to disrupt and lyse the proteins, incubate it for 5 minutes at room temperature, then centrifuge the lysate at 12,000 × g for 2 minutes at room temperature.  4. Transfer the supernatant to a clean 1.5 ml RNase–free micro centrifuge tube.  5. Add 200μl 100% ethanol to the microcentrifuge tube. Disperse any precipitate by vortexing or pipetting up and down several times (use RNase-free pipet tips).  6. Transfer the sample (including any remaining precipitate) to the Spin Cartridge (with a Collection Tube). Centrifuge at 12,000 × g for 15-30 seconds at room temperature. Discard the flow through. Optional: If your downstream application requires DNA-free total RNA, proceed to On–Column DNase Treatment during RNA Purification at this time.  7. Add 500 μl Buffer RBF2 to the Spin Cartridge. Centrifuge at 12,000 × g for 15 -30 seconds at room temperature. Discard the flow-through and the Collection Tube. Place the Spin Cartridge into a new Collection Tube. 8. Add 500μl Wash Buffer RBFW with ethanol to the Spin Cartridge.  9. Centrifuge at 12,000 × g for 15 seconds at room temperature. Discard the flow-through, and reinsert the Spin Cartridge in the same Collection Tube.  10. Repeat Steps 8–9 once again. Centrifuge again for 1 minute with the empty spin basket to remove residue of wash solution. 11. Carefully place spin filter in a new clean tube, add 20-50 μl Elution Buffer RBFE (RNase-free water) to the middle of the spin filter membrane. Incubate at room temperature for 1 min. Centrifuge for 1 min. at 12,000 x g.  12. Discard spin filter. RNA in the tube is now application ready.
 
 
Hints and Troubleshooting Guide
 
Concentrating the RNA Volume of RNA will be 50 μl. If this is too dilute for your purposes, add 5 μl of 3M NaAcetate and mix. Then add 150 μl of 100% cold ethanol. Mix, and incubate at -70°C for 15 minutes or -20°C for 2 hours to overnight. Centrifuge at 10,000 x g for 10 minutes at 4°C. Decant all
GenQuikTM Body Fluid RNA Extraction Kit                                                                 Page | 6
 
 Tel.: 630-916-0225 Fax: 630-916-0250 www.orochem.com Orochem Technologies Inc 331 Eisenhower Ln South, Lombard, IL 60148
liquid. Briefly dry residual ethanol in a speed vacuum or ambient air. Do not over-dry pellet or re-suspension may be difficult. Resuspend precipitated RNA in desired volume of RNase-Free water.
 
Storing RNA
 
RNA is eluted in RNase-Free water and should be used immediately or stored at -20°C or -80°C to avoid degradation. RNA can be precipitated in Ethanol and stored at -20°C to ensure minimal degradation during long term storage.
 
DNase Digestion
 
This kit will isolate RNA that in most cases is free of genomic DNA. However, if you plan to do reverse transcription reactions or RT PCR, we highly recommend that you treat your RNA with DNase prior to proceeding.
 
For Body Fluid RNA Extraction in 96-well formats, please call Orochem Technologies at 630-916-0225.