AURA PURE
RNA ISOLATION KIT
MNP-R001
Aura Biotechnologies Private Limited
Survey No: 270/1, Plot No: 1&2,
Galaxy Road, Ayanambakkam, Chennai-600095, India Phone: +91-44-49595785 Email: [email protected]
www.aurabiotech.com
PRINCIPLE:
The AURA PURE RNA Isolation kit is designed for the isolation of RNA from cell-free body fluids such as serum or plasma, blood, homogenized tissue sample suspensions, stool sample suspensions, swab washes and any other mammalian cells. This kit provides reagents and magnetic beads for isolation of 100 samples. The procedure is based on the adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. Sample lysis is achieved by incubation with a Lysis Buffer ARL1 containing chaotropic ions digestion. For binding of RNA to the paramagnetic beads, Binding Buffer ARB2 and the Aura Pure Beads are added to the lysate. After magnetic separation, the paramagnetic beads are washed to remove contaminants and salts using Wash Buffers ARW3, ARW4, and 80 % ethanol. Residual ethanol from previous wash steps is removed by air drying.
Finally, highly pure RNA is eluted with low-salt elution buffer or nuclease free water. Purified RNA can directly be used for downstream applications. The Aura Pure RNA Isolation kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators.
KIT SPECIFICATIONS:
Aura Pure RNA Isolation Kit is designed for rapid manual and automated small-scale preparation of RNA from various types of clinical samples. The kit is designed for use with Aura Pure magnetic separator which can hold 12 microcentrifuge tubes for the easy isolation of 12 RNA samples about 90 minutes. And this kit can be easily adoptable in any 96-well magnetic separator plate or other automated magnetic separation systems. The purified RNA and DNA can be used directly as template for RT-PCR, PCR, or any kind of enzymatic reactions.
KIT CONTENT
Kit contents For 50 Preparations For 250 Preparations
Aura Pure Beads 2 ml 10 ml
Lysis Buffer 12 ml 60 ml
Binding Buffer 30 ml 150 ml
Wash Buffer 2 X 10 ml 2 X 50 ml
Elution Buffer 5 ml 25 ml
Dnase-I 1 No 5 X 1 No
Dnase Buffer 10 ml 50 ml
User manual 1 1
MATERIAL TO BE SUPPLIED BY USER
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety datasheets (MSDSs), available from the product supplier.
• 100% Ethanol
• Isopropanol
• Micropipettes (variable range)
• Micropippette tips
• Microcentrifuge Tubes
• Heating block or Water Bath
• Vortex Mixer
STORAGE
Aura Pure RNA Isolation Kit is stable for 12 months from the date of manufacture without showing any reduction in performance. All other kit contents can be stored at room temperature (15-25°C) Except Dnase-I. After adding the Dnase-1 into the buffer, store at 2-8°C for short term usage or -20°C for long term usage.
INTENDED USE
Aura Pure RNA Isolation Kit is intended for molecular biology applications. All due care and attention should be exercised in the handling of the products. We recommend all users of AURA’s products to adhere to the NIH guidelines or other standard guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.
SAFETY INFORMATION
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDS).
Emergency medical information in English, call:
Tel: +91 44 49595785
QUALITY CONTROL
In accordance with AURA’s ISO-certified Total Quality Management System, each lot of the Aura Pure RNA Isolation Kit is tested against predetermined specifications to ensure consistent product quality.
IMPORTANT INSTRUCTION
A. PREPARATION OF SAMPLE MATERIALS 1. Blood samples:
A sample volume of 100–200 μL blood is recommended. Do not use higher volumes. When processing less than 200 μL sample adjust with PBS buffer to a final volume of 200 μL.
2. Any Mammalian Cells:
Use approximately 1X106 cells for the RNA isolation. Using more cells may leads to poor yield and quality.
3. Tissue samples:
Homogenize tissue samples. Typically 5–10 mg sample material can be homogenized in 400 μL PBS buffer using a homogenizer. If necessary, higher amounts of sample material can be used (up to 25 mg). It should be considered that the as purified total RNA may cause inhibition in the subsequent PCR assays. Centrifuge the homogenized sample and use up to 200 μL clear supernatant for further processing. If using less than 200 μL adjust with PBS buffer to a final volume of 200 μL.
4. For isolation of viral RNA from tissue:
Tissue can also be disrupted in a buffer containing chaotropic salt (e.g., Lysis buffer) and beta-mercaptoethanol or TCEP reducing agent.
5. Swab samples
Incubate the swabs in Lysis Buffer for 10-20 min with agitation. Then remove the swab pressing it against the walls of the tube to squeeze out most of the liquid.
6. Feces
Mix 1 volume of feces (e.g., 500 μL) with an equal volume of PBS buffer. Mix vigorously by vortexing for 1 min. Allow the particles to settle down or centrifuge with low speed (e.g., at 500 x g). Proceed with the cleared supernatant.
7. Clean up of TRIZOL purified samples
After phase separation by centrifugation proceed with the aqueous phase (the colorless upper phase; Approximately 400 μL). For further processing start with step 2 of the purification protocol by mixing 400 μL of the aqueous phase with 600 μL binding Buffer and 40 μL AURA PURE-Beads.
1. Read the entire procedure carefully before starting the experiment.
2. Use fresh tip while adding different solution to the tube.
B. PREPARATION OF BUFFERS
• Wash Buffer: Add 40 ml ethanol into 10 ml of buffer and store at room temperature with tightly closed cap.
• Dnase Reaction Mix: Add the entire Dnase into the reaction mix (Dnase Buffer, DB) and solubilize completely. Make it as aliquate and store at -20°C for long term storage.
• All other buffers are delivered ready-to-use.
B. RNA ISOLATION PROTOCOL
This protocol is designed for isolation of RNA can be performed in reaction tubes with suitable magnetic separators. This protocol is for manual use and serves as a guideline for adapting the kit to robotic instruments.
1. Add 100 μL of sample to a suitable reaction tube and add 250 μL Lysis Buffer to the reaction tube. Mix well by vortexing and incubate at room temp for 5 min.
2. Add 350 μl of isopropanol into the lysate and mix well by vortexing. Incubate the lysate mixture for 5 min at room temp.
Note (Optional): Mix by pipetting up and down 6 times and shake for 5 min at room temperature.
3. Add 40 μL resuspended AURA PURE Beads and mix well by gently by inverting the tube up and down and incubate for 5 min at room temp.
Note (Optional): Mix by pipetting up and down 6 times and shake for 5 min at room temperature.
4. Separate the magnetic beads by placing the tubes into a magnetic separator. Wait at least 1 min for all beads have been attracted to the magnets. Remove and discard supernatant by pipetting without disturbing the magnetic beads.
5. Remove the microcentrifuge tube from the magnetic separator and add 400 μl wash buffer. Shake at RT for 15 sec.
6. Settle the magnetic beads on a magnetic stand and discard the supernatant. Repeat the wash step by adding wash buffer for the total of 2 washes.
7. After 3rd wash remove the supernatant completely and air dry the tubes at room temp for ~5 min.
8. Add 150 μl of DNase reaction mix and mix well by vortexing for 15 sec at RT, centrifuge shortly and incubate for 10-15 min at 37 °C.
9. Add 600 μL Buffer Binding Buffer and resuspend the beads by shaking until the beads are resuspended completely (1–3 min). Alternatively, resuspend beads completely by repeated pipetting up and down and incubate for 10 min at room temp.
10. Settle the magnetic beads on a magnetic stand and discard the supernatant. Add 400 μl wash buffer Wash Buffer and Shake at RT for 15 sec.
11. Settle the magnetic beads on a magnetic stand and discard the supernatant. Repeat the wash step by adding ARW3 for the total of 2 washes.
12. After completely removing the wash buffer, Air dry the magnetic bead pellet for ~5 min at room temperature.
13. Add 40 μl of Elution Buffer to the magnetic beads to elute RNA. Resuspend the magnetic beads completely and Incubate for 3-5 min.
14. Settle the magnetic beads on a magnetic stand for at least 2 min and carefully collect the RNA elute into fresh tube and store at -80°C or below or use it immediately.
TROUBLESHOOTING:
Problem Possible cause and suggestions
Poor yield / low sensitivity
Incomplete sample lysis
• Sample mixed with Lysis Buffer and was not thoroughly homogenized. The mixture has to be shaken continuously.
Alternatively, prolong incubation time with lysis buffer.
Insufficient elution buffer volume
• Bead pellet must be covered completely with elution buffer and needs to be fully resuspended.
Insufficient performance of elution buffer during elution step
• Remove all buffer completely from the bead pellet after the binding and wash steps. Remaining buffer decreases the efficiency of the subsequent steps.
Aspiration of attracted bead pellet
• Do not disturb the attracted beads while aspirating the supernatant. This requires special caution when removing the lysate from the beads as the lysate is usually too opaque to allow visual control of the pellet.
Aspiration and loss of beads
• Time for magnetic separation too short or aspiration speed too high.
Low purity / low sensitivity
Insufficient washing procedure
• Use only the appropriate combinations of separator and plate, for example, AURA PURE Magnetic separator.
• Make sure that beads are resuspended completely during the washing procedure. If shaking is not sufficient to resuspend the beads completely mix by repeated pipetting up and down.
Poor performance of RNA in downstream
applications
Carry-over of ethanol from wash buffers
• Be sure to remove all of the 80 % ethanolic wash solution from the final wash, as residual ethanol interferes with downstream applications.
Ethanol evaporation from wash buffers
• Close buffer bottles tightly, avoid ethanol evaporation from buffer bottles as well as from buffer filled in reservoirs. Do not reuse buffers from buffer reservoirs.
Carry-over of beads
Time for magnetic separation too short
• Increase separation time to allow the beads to be completely attracted to the magnetic pins before aspirating any liquid from the well.
Aspiration speed too high (elution step)
• High aspiration speed during the elution step may cause bead carry-over. Reduce aspiration speed for elution step.
For More Details, Contact:
Aura Biotechnologies Private Limited
Survey No: 270/1, Plot No: 1&2, Galaxy Road, Ayanambakkam, Chennai-600095, India
Phone: +91-44-49595785 Email: [email protected] www.aurabiotech.com