Surface Bind RNA Purification

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A whole new approach to RNA purifcation using Surface Bind tecnology

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he Surface Bind RNA Purification Kit is designed for the optimal purification of high-quality RNA from total RNA or enzymatic reactions, such as in vitro transcription. Based on Solid Surface Reversible Binding (SSRB) technology, this purification system utilizes micro-tube coated with proprietary turbo binders acting to selectively capture and efficiently bind RNA from reaction mixtures. No spin-column, filter plates, silica membranes, or magnetic beads are needed for cleanup, also no vacuum or filtration steps are required in the purification process.

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he components included with Surface Bind RNA Purification Kit are listed below. Upon receipt, store all components at room temperature.

Box 1 | Contents
Kit Contents
# of Purifications ABP-PP-SBRNA025 ABP-PP-SBRNA050

25 25 1.0 mL 1.0 mL 2.5 mL 2.2 mL

50 50 2.0 mL 2.0 mL 5.0 mL 4.4 mL

BT5:

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n the presence of Binding Buffer, RNA molecules (ssRNA > 60 bases, dsRNA > 100 bps), including RNAs from in vitro transcription reactions, capped RNAs, amino allyl-modified RNAs, Biotin and Cy-dye labeled RNAs can specifically interact with the turbo binders and bind to the tubes while proteins, nucleotides, short oligo nucleotides, salts and other contaminants will remain in the solution. Unbound material is then removed in subsequent washing steps. The purified RNA is easily eluted in 10 mM Tris Elution Buffer or water allowing for downstream applications such as RT and other gene expression analysis directly in the same tube without sample elution.

Binding Tubes

RB7:

Binding Buffer

WBlR: WB2: EB3:

Washing Buffer IR Washing Buffer II Elution Buffer

Additional Materials Needed •100% Ethanol and Isopropanol (ACS grade or better) •Single-channel pipettor and RNase-free tips •Benchtop Centrifuge

Features
♦ Easy to handle: All procedures have been optimized with single tube for ease-of-use. You can perform RNA purification and downstream assays all in the same tube without sample transferring. ♦Maximum recovery: Capable of maximizing RNA recovery from original RNA sample is the unique feature of the kit. Unlike conventional silica membrane or bead based purification platform, there are no void spaces in the Binding Tube. This novel solid-surface capturing technology prevents washing buffer carryover and sample trapping problems associated with membrane or bead-based column/plate purification methods. This feature not only allows fast buffer washing and easy sample elution, but also provides maximum sample capture and release. RNA molecules as few as 100 ng can be efficiently isolated with over 80% recovery efficiency. ♦ Technical data: RNA length of ssRNA > 60 bases or dsRNA > 100 bps is recommended for the procedure. RNA input from 100 ng to 500 μg can be purified with each purification.

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

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Preparation
WBIR: For ABP-PP-SBRNA025 kit, add 4.0 ml of
100% ethanol to Washing Buffer IR (WBIR) and mix well. For ABP-PP-SBRNA050 kit, add 8.0 ml of 100% ethanol to Washing Buffer IR (WBIR) and mix well. Mark bottle that ethanol has been added. ♦ Store at 4 ° C and use Washing Buffer (WBIR) containing ethanol within six (6) months. Be sure not to scrape the walls of the tube with pipette tips during aspiration as the products are bound to the walls of the Binding Tube. Washing RNA Products 1. Add 150 μl Wash Buffer IR (WB I R) containing ethanol to each tube. Mix by pi petting solution up and down 4-5 times. 2. Remove the Wash Buffer IR (WBIR) from the Binding Tube using the methods suggested in step 6 of “Binding RNA Products”. 3. Add 200 l Wash Buffer II (WB2) containing ethanol to each well. Mix by pipetting solution up and down 4-5 times. 4. Remove the Wash Buffer II (WB2) from the Binding Tube using the methods suggested in step 6 of “Binding RNA Products”. 5. Repeat step 3 and 4 above for a total of two washes with Washing Buffer II (WB2). 6. After the final wash, to ensure complete removal of Washing Buffer, you may spin the Binding Tube very briefly after removing the majority of solution and aspirate the last drop of liquid at the bottom center of the tube with a pipet tip; or you may invert the tube and tap the tube on a stack of clean, absorbent paper gently and air-dry the Binding Tube in a lab hood for 8 - I 0 minutes to remove any residual liquid. Alternatively, put the tube in a 62°C air incubator or a hot blocker and air-dry the tube without cover for 2-4 minutes. Eluting RNA Products RNA attached to the walls of the Binding Tube can be stored directly in the well at -80 °C for long-term storage until further use. If the RNA is to be eluted, follow the procedure below. 1. Add 50- 80 μl Elution Buffer (EB3) into each tube. 2. Close the tube and vortex the tube for 10-30 seconds, or tap the Binding Tube (BT5) 10-15 times with a finger to allow the Elution Buffer (EB3) contacting the wall of the bottom of the Binding Tube. 3. Briefly centrifuge the tube to collect all solution, and place the tube on ice for downstream applications. If you are not going to analyze the samples immediately, store the tube at -80 °C until use.

WB2: For ABP-PP-SBRNA025 kit, add 10 ml of 100%

ethanol to Washing Buffer II (WB2) and mix well. For ABP-PP-SBRNA050 kit, add 20 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.

RB7: Prepare fresh working Binding Buffer (RB7)

each time prior to performing RNA isolation procedure based on the number of samples processed. To make fresh working Binding Buffer (RB7), for each 10 μl of Binding Buffer (RB7) add 40 μl of 100% isopropanol. Mix well. Prepare a master working Binding Buffer solution based on (1) the number of samples processed and (2) any anticipated loss, generally 10 %, during dispensing. Dispense 100 μl Binding Buffer containing isopropanol per 50-μl RNA sample. Discard the unused Binding Buffer at the end of the day. ♦ If desired, you may add an internal control into the purification procedure. Internal control RNA should be added together with the binding buffer.

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

Binding RNA Products 1. Transfer 50 μl RNA reaction solutions to a Binding Tube (BT5). (If sample is not exactly 50 l, adjust all reagents proportionately throughout the procedure) 2. Add 100 μl fresh working Binding Buffer (RB7) containing isopropanol to the Binding Tube (BT5). Mix well with the RNA sample by pipetting up and down the solution 10 times to obtain a homogenous solution. 3. Close the Binding Tube (BT5) cap. 4. Centrifuge the Binding Tube at maximum speed (about 13000 rpm or 16000 X g) at room temperature for 8 minutes to bind the RNA. 5. Open the cap. 6. Remove the solution by aspirating the solution from the exact center of the tube bottom with a pipette tip.

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 μl 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 Solution Check original RNA with gel electrophoresis or Agilent 2100 OR Add additional RNA sample. Mix RNA sample with fresh prepared RNA Binding Buffer (RB7) thoroughly before centrifuge. Make sure the Binding Tube was spun at 16000 x g for 8 minutes. If lower speed is used, increase the spin time.

Low centrifugation forces

Low product yield

Maneuver the pipette tip to the exact Pipette tips scrape walls of center of the bottom of the Binding Tube the Binding Tube too much during aspiration. If possible, use a narduring aspiration of solution row gel-loading pipette tip for aspiration to reduce the scrapping. 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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