RNA Purification System,

Employs modified silica membranes as selective nucleic acid captures in a spin-column format.

he Allele RNA Purification Kit is designed for the purification of high-quality RNA from total RNA or RNA enzymatic reactions, such as in vitro transcription. This system employs modified silica membranes as selective nucleic acid captures in a spin-column format. RNA molecules (ssRNA > 80 bases, dsRNA > 150 bps), including RNAs from in vitro transcription reaction, capped RNAs, amino allyl-modified RNAs, Biotin and Cy-dye labeled RNAs can specifically interact with the modified silica membrane in the RNA Binding Buffer, while nucleotides, short oligonucleotides, salts, proteins and other contaminants are not bound and remain in the solution which is passed through. After washing the column with washing buffer to remove unbound material, the purified RNA can be easily eluted in 10 mM Tris Elution Buffer or water. Purified RNA can be used for any downstream application that requires high quality RNA, such as qRT-PCR, microarray, RNA-seq, RNase protection assay and other transcriptional RNA analysis.

Features
Fast and Easy Protocol: The procedure consists of three steps: RNA binding to membrane, contaminant being washed away and RNA elution from membrane. It takes less than 15 minutes to finish the purification procedure. High Yield and Quality: The modified membrane and RNA binding buffer system provides for maximum RNA capturing and release. The recovery is more than 75% of input RNA of high purity with efficient removal ofnucleotides, short oligonucleotides, salts and proteins. Technical data: RNA length of ssRNA > 80 bases or dsRNA > 150 bps is recommended for the procedure. RNA input from 1 g to 300 g can be purified with each purification.

General Precautions
Box 1 | Contents
Kit Contents
# of Purifications ABP-PP-RNAPUR025 ABP-PP-RNAPUR050

This kit is for research use only. All due care and attention should be exercised in the handling of the kits.
25 25 50 50

CC1:

Spin-Columns with Collection Tubes

RT1:

Recovery Tubes

25 5.0 mL 5.0 mL 6.25 mL

50 10 mL 10 mL 12.5 mL

Wear a laboratory coat, disposable gloves, and eye protection when handling reagents and tubes. Avoid ingestion and inhalation of reagents. In case of contact, wash thoroughly with water. See Material Safety Data Sheets (MSDS) for emergency procedures in case of accidental contact or ingestion. MSDS information is available upon request. Always use proper aseptic techniques to avoid nuclease contamination when working with RNA. Use only sterile, new pipette tips to prevent cross contamination.

RB4:

RNA Binding Buffer

WB2: EB3:

Washing Buffer II Elution Buffer

Preparation
WB2: For ABP-PP-RNAPUR025 kit, add 20 ml of
100% ethanol to Washing Buffer II (WB2) and mix well. For ABP-PP-RNAPUR050 kit, add 40 ml of 100% ethanol to Washing Buffer II (WB2) and mix well. Mark bottle that ethanol has been added. Store at room temperature and use Washing Buffer II (WB2) containing ethanol within six (6) months. Avoid nuclease contamination and clean lab bench with RNase inactivating agents such as RNase-Off or RNase-Away and wipe with 75%ethanol. Set a water baths or heat blocks at 94 C.

Additional Materials Needed 100% Ethanol (ACS grade or better) Pipettor and RNase-free tips Benchtop Centrifuge and Vortexer Nuclease-free 1. 7 ml microcentrifuge tubes Heating block or water bath

he components included with RNA Purification System are listed above. Upon receipt, store all components at room temperature with the exception of RNA Binding Buffer (RB4), which needs to be stored at 4 C..

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The protocol below is for the purification of RNA from 50 l reaction. The purification procedure may be scaled from 10100 l by proportionately adjusting all reagents throughout the procedure.

Protocols
Binding RNA to Spin Column 1. Bring the RNA sample to 50l with Elution Buffer (EB3) and mix well. 2. Add 170l RNA Binding Buffer (RB4) to the sample and mix well by pi petting solution up and down 7- 8 times to yield a homogenous solution. 3. Add 130l 100% ethanol to the sample and mix well by pipetting solution up and down 7-8 times to yield a homogenous solution. 4. Transfer 350l of the sample solution from step above to a Spin-Column with a Collection Tube (CCI). 5. Centrifuge the Spin-Column at 8000 x g for one minute at room temperature. Discard the filtrate in the Collection Tube and reinsert the Spin-Column back into the Collection Tube. Washing RNA Products 1. Add 400 l of Wash Buffer II (WB2) with ethanol to the Column. 2. Centrifuge the Spin-Column at 8000 g for 1 minute at room temperature. Discard the filtrate. 3. Repeat step 1 and 2 above for a total of two washes. Discard the filtrate. 4. Centrifuge the spin-column at maximum speed (10000- 15000 g, or 10000- 14000 rpm) for extra 1 minute to remove any residual wash buffer with ethanol. Discard Collection Tube. 5. Reinsert the Spin-Column into a new clean microcentrifuge Recovery Tube (RTI). Eluting RNA Products 1. Preheat 200-1000l Elution Buffer (EB3) in a 1.7 ml tube to 94 C with a heat block or a water bath. 2. Add 50l pre-heated Elution Buffer (EB3) into the center of the Spin-Column. 3. Incubate the column for 1 minute at room temperature. 4. Centrifuge the column at maximum speed for 1 minute at room temperature. The Recovery Tube (RTI) contains purified RNA.

5. To maximize RNA recovery, you may perform a second elution with 50l pre-heated Elution Buffer (EB3) with the same recovery tube, if desired. This can increase the total RNA yield by 10-30 %, but the final concentration of purified RNA in the recovery tube is reduced because of the increased volume. 6. Store the purified RNA on ice. At this point, samples are ready for desired downstream application such as qRT-PCR, or store the RNA samples at -80 C for long-term storage until further use.

Electrophoresis and Downstream Application Purified RNA can be examined by agarose gel electrophoresis. Yield can be measured with a spectrophotometer at 260 nm, fluorescent RNA assays or other quantification methods by diluting an aliquot of the purified RNA sample (usually 1:50- 1:200 dilution). For electrophoresis, loading 3-8 l purified RNA is recommended. The purified RNA is suitable for use in qRTPCR, microarray, RNA-seq, RNase protection assay and other transcriptional RNA analysis.

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Box 2 | Troubleshooting Problem Cause Amount of RNA used was less than recommended or poor quality of starting material Incomplete Mixing Low product yield Solution Check original RNA with gel electrophoresis or Agilent 2100 OR Add additional RNA sample. Mix RNA sample with RNA Binding Buffer and 100% Ethanol thoroughly before loading onto spin-column Make sure lab bench and pipettes are clean and RNase-free. Wear gloves at all times during the whole procedure and change gloves frequently to protect reagents and RNA from nuclease that are present on skin. Use RNase-free pipette tips and tubes to handle all solutions; avoid used tips or used tubes for reagents Be sure to add all components. Check positive and negative controls for RTPCR reaction.

Nuclease contamination

No RT-PCR product

Missing Component in the RT-PCR mixture

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