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The PureLink miRNA Isolation Kit provides a rapid and efficient method to purify small RNA molecules from biological samples for functional analysis. The kit is specifically designed to isolate high-quality small RNA molecules including tRNA, 5S rRNA, 5.8S rRNA and regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA). Using the kit to isolate small RNA molecules results in efficient removal of large RNA molecules such as 28S rRNA, 18S rRNA, and mRNA that can inhibit expression analysis of small RNA molecules. See below for an overview of the purification protocol. The purified RNA is suitable for northern blot analysis and microarray analysis.
The PureLink miRNA Isolation Kit is based on the selective binding of small RNA molecules to silica-based membrane in the presence of 70% ethanol. The lysate is prepared from mammalian cells and tissue, plant tissues, E. coli cells and yeast cells using Binding Buffer (L3) containing guanidine isothiocyanate, a chaotrope capable of protecting the RNA from endogenous RNases. Ethanol is added to the lysate to a final concentration of 35% and the lysate is processed through the first Spin Cartridge. Large RNA molecules bind to the silica-based membrane in the cartridge while small RNA molecules are recovered in the flow through fraction. Ethanol is added to the flow through fraction to a final concentration of 70% and the sample is loaded onto a second Spin Cartridge. The small RNA molecules bind to the silica-based membrane in the cartridge and impurities are removed by thorough washing with Wash Buffer. The RNA is then eluted in sterile, RNase free water.
Small regulatory RNA molecules play an important role in regulation of gene expression in various organisms by binding to the target mRNAs through sequence complementation (Liu, 2004). Small RNA molecules include tRNA, 5S rRNA, and 5.8S rRNA and regulatory RNA molecules such as siRNA and miRNA. siRNAs are ~21-23 nucleotide double-stranded RNA molecules involved in post-transcriptional gene silencing using the RNAi (RNA interference) pathway (Elbashir et al., 2002). miRNAs are ~21-22 nucleotide double stranded RNA molecules that play important roles in the regulation of translation and degradation of target mRNAs through base pairing to partially complementary sites in the untranslated regions of the message (Lim, 2003). Isolation and purification of small regulatory RNA molecules allows elucidation of biologically significant pathways for regulation of gene expression and requires enrichment of small RNA molecules from biological samples for functional analysis. The standard protocols for isolating total RNA and mRNA are not optimized for isolation of small RNA molecules and result in the loss of substantial amounts of small RNA. Co-purification of larger RNA molecules such as 28S rRNA, 18S rRNA, and mRNA with small RNA inhibits expression analysis of small RNA. The PureLink miRNA Isolation Kit is specifically designed for purification of small RNA with minimal contamination from large RNA molecules.
Using PureLink miRNA Isolation Kit to isolate small RNA molecules provides the following advantages:
All components of the PureLink miRNA Isolation Kit are shipped at room temperature. Upon receipt, store all components at room temperature
The components included in the PureLink miRNA Isolation Kit are listed below. Sufficient reagents are provided in the kit to perform 25 reactions.
Component | Amount |
---|---|
Binding Buffer (L3) | 45 mL |
Wash Buffer (W5) | 10 mL |
Buffer (W4) for single column purification | 3 mL |
Sterile, RNase-free Water | 5 mL |
Spin Cartridges with Collection Tubes | 50 |
Wash Tubes (2.0 mL) | 50 |
Recovery Tubes (1.7 mL) | 50 |
The PureLink miRNA Isolation Kit is functionally qualified as described below. Yeast tRNA and 10 bp DNA Ladder are purified using the kit as described in this manual. The purified small RNA is analyzed by gel electrophoresis. Agarose gel electrophoresis must show the co-purification of yeast tRNA with 10 bp DNA Ladder fragments less than 40 bp and DNA fragments greater than 40 bp must not be detectable. In addition, each kit component is free of ribonuclease contamination and is lot qualified for optimal performance.
This quick reference sheet is included for experienced users of the PureLink miRNA Isolation Kit. If you are a first time user, follow the detailed protocol in this manual.
Mammalian Cells (up to 1 x 106 cells) |
1. Harvest cells and add 300 µL Binding Buffer (L3) to cells. |
2. Mix well by vortexing and add 300 µL 70% ethanol. Mix well |
Mammalian Tissue (up to 5 mg) |
1. Add 300 µL Binding Buffer (L3) and homogenize tissue using tissue homogenizer |
2. Centrifuge at 12,000 x g for 1 minute to remove any particulate materials. Transfer supernatant to a clean tube and add 300 µL 70% ethanol. Mix well by vortexing |
Plant Tissue (up to 100 mg) |
1. Grind tissue to a powder in liquid nitrogen and add 300 µL Binding Buffer (L3). Mix well by vortexing. |
2. Centrifuge the lysate at 12,000 x g for 2 minutes to remove any particulate materials. Transfer supernatant to a clean tube and add 300 µL 70% ethanol. Mix well by vortexing. |
Yeast Cells (up to 2x 107 cells) |
1. Harvest 1 mL fresh, log phase yeast cells (OD660 ~ 1.0) by centrifugation and resuspend cell pellet in 70 µL TE Buffer. |
2. Add 30 units zymolase (lyticase) enzyme to the lysate and incubate for 30 minutes at 30 °C. |
3. Add 300 µL Binding Buffer (L3) and 210 µL of 96-100% ethanol. Mix well by vortexing. |
Bacterial Cells (up to 5x 106 cells) |
1. Harvest up to 5 x 106 E. coli cells by centrifugation and resuspend cell pellet in 90 µL TE with 1 mg lysozyme. Add 1 µL 5% SDS and mix well. |
2. Incubate at room temperature for 5 minutes. |
3. Add 300 µL Binding Buffer (L3) and add 210 µL 96-100% ethanol. Mix well by vortexing. |
The purification procedure is designed for use with a microcentrifuge capable of centrifuging >10,000 x g and can be completed in 10-15 minutes.
1. Add prepared sample from previous page to a Spin Cartridge in a collection tube. |
2. Centrifuge the Spin Cartridge at 12,000 × g for 1 minute. Total RNA is bound to the cartridge. Keep the flow through. |
3. Add 96-100% ethanol to the flow through to a final concentration of 70%. Mix well by vortexing. |
4. Transfer 700 µL sample to a second Spin Cartridge in collection tube. Centrifuge the Spin Cartridge at 12,000 x g for 1 minute. Small RNA molecules bind to the Spin Cartridge. Discard the flow through. |
5. Repeat Step 4 for the remaining sample. Place the Spin Cartridge in the collection tube. |
6. Wash the Spin Cartridge with 500 µL Wash Buffer (W5) with ethanol Centrifuge at 12,000 × g for 1 minute. Discard the flow through. |
7. Repeat wash step with 500 µL Wash Buffer (W5) once. |
8. Discard the collection tube and place the Spin Cartridge in a Wash Tube supplied with the kit. |
9. Centrifuge the Spin Cartridge at maximum speed for 1 minute to remove any residual Wash Buffer (W5). |
10. Place the Spin Cartridge in a clean 1.7-ml Recovery Tube supplied with the kit. |
11. Elute the RNA with 50-100 µL sterile RNase-free water supplied with the kit (add water to the center of the cartridge). |
12. Incubate at room temperature for 1 minute. |
13. Centrifuge the Spin Cartridge at maximum speed for 1 minute to elute RNA. The Recovery Tube contains purified small RNA molecules. Discard the Spin Cartridge. |
14. Store purified RNA at -80°C or use RNA for the desired downstream application. |
Instructions for preparing lysates from mammalian cells and tissues, plant tissues, yeast, and bacteria are described below. To obtain high-quality RNA, follow the guidelines recommended below. The PureLink miRNA Isolation Kit buffers contain guanidine isothiocyanate. Always wear a laboratory coat, disposable gloves, and eye protection when handling buffers. Do not add bleach or acidic solutions directly to solutions containing guanidine isothiocyanate or sample preparation waste as it forms reactive compounds and toxic gases when mixed with bleach or acids.
Observe the following guidelines to prevent RNase contamination:
There are different protocols for preparing lysates depending on the starting material (sample). Based on your sample, choose an appropriate lysate preparation protocol from the table below. To obtain high yield of small RNA molecules and minimize any degradation, collect the sample and proceed immediately to sample preparation or freeze the sample in liquid nitrogen immediately after collection. To obtain the best results, use the appropriate protocol based on your sample and the recommended sample amount for purification as described in the table below. If you wish to start with less amount of sample, use the appropriate protocol based on your sample without changing the volume of reagent used. Note that if you start with less amount of sample, the RNA yield may be lower.
Sample | Amount | Link to protocol |
---|---|---|
Mammalian cells | up to 1 x 106 cells | Mammalian cells lysate |
Mammalian tissues | up to 5 mg | Mammalian tissue lysate |
Plant tissues | up to 100 mg | Plant tissue lysate |
Yeast cells | 2 x 107 cells | Yeast lysate |
E. coli cells | 5 x 106 cells | E. coli lysate |
Dicing reaction | 50 µL | Dicing Reaction |
Larger sample amount | — | Large sample amount |
To minimize RNA degradation and to obtain optimal RNA yield, it is important to perform complete disruption of tissue in lysis buffer quickly and not to exceed the recommended starting amounts for various samples listed in the above table.
Using increased amount of starting material results in co-purification of large molecules such as 18S/28S rRNA, mRNA, and possibly gDNA that did not bind to the column during the first column screening, is retained as flow through, and co-purifies with small RNA molecules. If you wish to process large amount of starting material, see Large sample amount for recommended conditions.
Procedure to prepare lysate from mammalian cells is described below.
Procedure to prepare lysate from mammalian tissues is described below.
Procedure to prepare lysate from plant is described below.
Procedure to prepare lysate from yeast cells is described below.
Procedure to prepare E. coli cell lysate is described below.
If you wish to process large sample amount as compared to the amount listed on page 6, use TRIzol Reagent to prepare lysate as described below.
Tissues
Homogenize tissue samples in 1 mL TRIzol Reagent per 50-100 mg tissue using a tissue homogenizer.
Adherent cells
Lyse cells directly in a culture dish by adding 1 mL TRIzol Reagent to a culture dish and passing the cell lysate several times through a pipette. The amount of TRIzol Reagent required is based on the culture dish area (1 mL per 10 cm2) and not on the number of cells present.
Suspension cells
Harvest cells and pellet cells by centrifugation. Use 1 mL of the TRIzol Reagent per 5-10 × 106 animal, plant or yeast cells, or per 1 × 107 bacterial cells and lyse cells by repetitive pipetting.
Phase Separation
If you are purifying d-siRNA produced in the dicing reaction using the BLOCK-iT Dicer RNAi Kit (available from Invitrogen) or an equivalent kit, use the protocol below to prepare samples prior to purification. To 300 µL dicing reaction, add 300 µL Binding Buffer (L3) and 300 µL 96-100% ethanol to obtain a final volume of 900 µL. Mix well by vortexing. Proceed to the Binding Step.
Note: You can also use 300 µL isopropanol instead of ethanol as suggested in the BLOCK-iT Dicer RNAi Kit manual for preparing the dicing reaction for purifying d-siRNA.
The purification procedure is designed for purifying small RNA molecules using spin column based centrifugation in a total time of 10-15 minutes.
The purification procedure described in this section is designed for purifying small RNA molecules from tissues, cells, and dicing reaction using the two-column protocol. If you are purifying small RNA molecules from a dicing reaction, an optional one-column protocol followed by ethanol precipitation of RNA is described on above. If you wish to purify total RNA from the same sample, the total RNA is bound to the first spin column and can be washed and eluted using the reagents and protocol described in the PureLink Micro-to-Midi Total RNA Purification Kit.
For two-column purification protocol, use sterile, RNase-free water supplied with the kit to elute small RNA molecules. For one-column purification protocol, use Buffer (W4) for single column purification supplied with the kit to elute small RNA molecules.
Add 40 mL 96-100% ethanol to 10 mL Wash Buffer (W5) included with the kit. Store the Wash Buffer (W5) with ethanol at room temperature.
The quantity of the purified small RNA molecules is easilyquantitated using UV absorbance at 260 nm or Quant-iT RNA Assay Kit.
Quant-iT RNA Assay Kits
The Quant-iT RNA Assay Kit provides a rapid, sensitive, and specific method for RNA quantitation with minimal interference from DNA, protein, or other common contaminants that affect UV absorbance readings. The kit contains a state-of-the-art quantitation reagent and pre-diluted standards for standard curve. The assay is performed in a microtiter plate format and is designed for reading in standard fluorescent microplate readers.
Typically, small RNA molecules isolated using the PureLink miRNA Isolation Kit have an OD260/280 of >1.8 when samples are diluted in Tris-HCl (pH 7.5). An OD260/280 of >1.8 indicates that RNA is reasonably clean of proteins and other UV chromophores (heme, chlorophyl, etc.) that could either interfere with downstream applications or negatively affect the stability of the stored RNA. To visualize small RNA molecules, perform denaturing gel electrophoresis using 10% or 15% TBE acrylamide gels. An example of denaturing TBE gel analysis is shown below.
Small RNA molecules from various samples were isolated using the PureLink miRNA Isolation Kit as described on this Web page. Samples (10 µL eluate) were analyzed on a Novex TBE-Urea 15% Gel and RNA bands were visualized with ethidium bromide staining after electrophoresis.
The yield of small RNA molecules obtained from various samples using the PureLink miRNA Isolation Kit is listed below. The RNA quantitation was performed with the Quant-iT RNA Assay Kit.
Material | Amount | Yield (μg) |
---|---|---|
HeLa cells | 1x106 | 1.29 |
293F cells | 1x106 | 1.95 |
Mouse liver | 5 mg | 0.71 |
Rat Spleen | 5 mg | 1.39 |
Spinach | 60 mg | 1.64 |
Yeast (S. cerevisiae) | 1x106 | 6.21 |
Bacteria (E. coli) | 2x106 | 0.55 |
Problem | Cause | Solution |
---|---|---|
Low RNA yield | Incomplete lysis or too much sample has clogged the filter |
|
Low RNA content | Various tissues have different small RNA content and some tissue may not contain any small RNA at detectable levels. | |
Flow through from first cartridge discarded | Do not discard the flow through from the first cartridge. The flow through contains small RNA molecules. | |
Incorrect binding conditions | For efficient binding of small RNA molecules, always add ethanol to the flow through to a final concentration of 70%. | |
Ethanol not added to Wash Buffer (W5) | Be sure to add 96–100% ethanol to Wash Buffer (W5) | |
Incorrect elution conditions |
| |
RNA quantitation performed with water | Be sure the RNA quantitation using UV absorbance is performed with 10 mM Tris-HCl, pH 7.0. to accurately measure the UV absorbance | |
RNA degraded | RNA contaminated with RNase | Follow the beginning guidelines to prevent RNase contamination. |
Poor quality starting materials | Always use fresh samples or samples frozen at -80°C. For lysis, process the sample quickly to avoid degradation. | |
Genomic DNA contamination | Large sample amount loaded on the first column |
|
Total RNA contamination | Processed large amount of starting material without using TRIzol Reagent |
|
25-0753 Version C 18-Jul-2005