PureLink miRNA Isolation Kit

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.

System overview

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 RNA Molecules

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.

Advantages

Using PureLink miRNA Isolation Kit to isolate small RNA molecules provides the following advantages:

• Rapid and efficient purification of high-quality small RNA molecules using spin column-based centrifugation
• Specifically designed to purify small RNA molecules including miRNA and siRNA from a variety of samples in less than 15 minutes
• Minimal contamination from large RNA molecules and genomic DNA
• Reliable performance of the purified small RNA in downstream applications

Shipping and storage

All components of the PureLink miRNA Isolation Kit are shipped at room temperature. Upon receipt, store all components at room temperature

Contents

The components included in the PureLink miRNA Isolation Kit are listed below. Sufficient reagents are provided in the kit to perform 25 reactions.

Product qualification

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.

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Introduction

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.

Sample preparation

Purification procedure

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.

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Preparing lysates

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.

General handling of RNA

Observe the following guidelines to prevent RNase contamination:

• Use disposable, individually wrapped, sterile plastic ware
• Use only sterile, new pipette tips and microcentrifuge tubes
• Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin
• Always use proper microbiological aseptic techniques when working with RNA
• Use RNase AWAY Reagent to remove RNase contamination from surfaces

Sample amount and quality

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.

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.

Materials needed

• 96–100% ethanol
• 70% ethanol
• Sample
• Binding Buffer (L3) supplied with the kit
• TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
• Zymolase (lyticase) enzyme for yeast lysate
• Lysozyme and 5% SDS for bacterial cell lysate
• Invitrogen Homogenizer (clarifier; for viscous or debris-containing samples)
• General tissue homogenizer for homogenizing mammalian and plant tissues
• TRIzol Reagent and chloroform for processing large sample amount
• Sterile, DNase-free microcentrifuge tubes
• Water bath or heat block set at 30°C for yeast lysate

Mammalian cells lysate

Procedure to prepare lysate from mammalian cells is described below.

1. For adherent cells (up to 1 x 10⁶ cells), remove the growth medium from the culture plate. For suspension cells (up to 1 x 10⁶ cells), harvest the cells and centrifuge the cells at 250 x g for 5 minutes to pellet cells. Remove the growth medium.
2. Resuspend cells from Step 1 in 300 µL Binding Buffer (L3) supplied with the kit. Mix well by vortexing or pipetting up and down until the cells appear lysed.
3. Add 300 µL 70% ethanol to the cell lysate. Mix well by vortexing.
4. Proceed to the Binding Step

Mammalian tissue lysate

Procedure to prepare lysate from mammalian tissues is described below.

1. Place ~5 mg of minced mammalian tissue into a sterile microcentrifuge tube.
2. Add 300 µL Binding Buffer (L3) supplied with the kit. Ensure the tissue is completely immersed in the buffer.
3. Homogenize the tissue using a tissue homogenizer.
4. Centrifuge the lysate at 12,000 x g for 5 minutes at room temperature to remove any particulate material.
5. Transfer the supernatant to a new, sterile microcentrifuge tube.  Note: If the lysate is viscous or contains cell debris, clarify the lysate using the Homogenizer available from Invitrogen (page viii).
6. Add 300 µL 70% ethanol to the lysate. Mix well by vortexing.
7. Proceed to the Binding Step

Plant tissue lysate

Procedure to prepare lysate from plant is described below.

1. Freeze the plant tissue in liquid nitrogen and grind the tissue to a powder. For soft tissue, cut the tissue into small pieces.
2. Add 300 µL Binding Buffer (L3) supplied with the kit to the tissue. For ground powder, vortex the powder until the powder is completely resuspended. For the soft tissue, homogenize using a tissue homogenizer.
3. Centrifuge the lysate at 12,000 x g for 2 minutes at room temperature to remove any particulate material.
4. Transfer the supernatant to another sterile microcentrifuge tube. Note: If the lysate is viscous or contains cell debris, clarify the lysate using a Homogenizer available from Invitrogen.
5. Add 300 µL 70% ethanol to the lysate. Mix well by vortexing.
6. Proceed to the Binding Step

Yeast lysate

Procedure to prepare lysate from yeast cells is described below.

1. Harvest 1 mL fresh, log-phase yeast cells (OD660 = 1.04) by centrifugation. If you are using a frozen cell pellet, proceed to Step 2.
2. Resuspend the cell pellet in 70 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
3. Add 30 units zymolase (lyticase) enzyme and incubate at 30°C for 30 minutes to lyse the cells.
4. Centrifuge at 12,000 x g for 5 minutes at room temperature to remove any particulate material.
5. Resuspend the pellet in 300 µL Binding Buffer (L3) supplied with the kit.
6. Add 210 µL 96-100% ethanol to the lysate. Mix well by vortexing.
7. Proceed to Binding Step

E. coli Lysate

Procedure to prepare E. coli cell lysate is described below.

1. Prepare 90 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 1 mg lysozyme enzyme. Store on ice until use.
2. Harvest up to 5 x 10⁶ E. coli cells by centrifugation. If you are using a frozen cell pellet, proceed to Step 3.
3. Resuspend the cell pellet in 90 µL TE buffer containing 1 mg lysozyme from Step 1.
4. Add 1 µL 5% SDS to the lysate and mix well by vortexing. Incubate at room temperature for 5 minutes.
5. Add 300 µL Binding Buffer (L3) supplied with the kit to the lysate. Mix well by vortexing.
6. Add 210 µL 96-100% ethanol to the lysate. Mix well by vortexing.
7. Proceed to the Binding Step

Large sample amount

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.

Lysate Preparation

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

1. Incubate the lysate with TRIzol Reagent at room temperature for 5 minutes to allow complete dissociation of nucleoprotein complexes.
2. Add 0.2 mL chloroform per 1 mL TRIzol Reagent and shake the tube vigorously by hand for 15 seconds. Avoid vortexing the sample.
3. Incubate at room temperature for 2-3 minutes.
4. Centrifuge the sample at 12,000 x g for 15 minutes at 4°C. After centrifugation, the mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. The volume of aqueous phase is ~600 µL.
5. Transfer ~ 400 µL of the colorless, upper phase containing RNA to a fresh tube.
6. Add 96-100% ethanol into 400 µL of the aqueous phase to obtain a final concentration to 35% (e.g. add 215 µL of 96-100% ethanol) and mix well by vortexing.
7. Proceed to the Binding Step

Dicing reaction

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.

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Introduction

The purification procedure is designed for purifying small RNA molecules using spin column based centrifugation in a total time of 10-15 minutes.

Materials needed

• 96-100% ethanol
• Microcentrifuge capable of centrifuging >10,000 x g
• Components supplied with the kit
• Wash Buffer (W5)
• Sterile, RNase-free Water (pH>7.0)
• Spin Cartridge in Collection Tubes
• Wash Tubes and Recovery Tubes

Follow the recommendations below to obtain the best results:

• Perform all centrifugation steps at room temperature
• Be sure to add ethanol to a final concentration of 70% and transfer sample to a second column to isolate small RNA molecules
• Perform the recommended wash steps to obtain high-quality RNA
• Pipet water in the center of the Spin Cartridge and perform a 1 minute incubation

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.

Elution buffer

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.

Before starting

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.

Binding step

1. Remove a Spin Cartridge in a Collection Tube from the package.
2. Add the lysate with Binding Buffer (L3) and ethanol (total volume is 600 µL) prepared as described on above to the Spin Cartridge.
3. Centrifuge the Spin Cartridge 12,000 × g for 1 minute at room temperature.
4. Keep the flow through. Add 96-100% ethanol to the flow through to obtain a final concentration of 70% ethanol and mix well by vortexing. Note: The lysate already contains 35% ethanol added during sample preparation.  For example: To 600 µL lysate, add 700 µL 96-100% ethanol and mix well by vortexing.  Note: To isolate total RNA from the same sample, place the Spin Cartridge into a new Wash Tube and process the Spin Cartridge for isolating total RNA as described in the PureLink Micro-to-Midi Total RNA Purification Kit
5. Remove another Spin Cartridge in a Collection Tube from the package and transfer 700 µL sample from Step 4 to the Spin Cartridge.
6. Centrifuge the Spin Cartridge at 12,000 × g for 1 minute at room temperature.
7. Transfer remaining sample from Step 4 to the Spin Cartridge from Step 6 and centrifuge at 12,000 x g for 1 minute at room temperature.
8. Discard the flow through and place the Spin Cartridge into the collection tube.
9. Proceed to the Washing Step, below.

Washing step

1. Add 500 µL Wash Buffer (W5) with ethanol (page 13) to the Spin Cartridge.
2. Centrifuge 12,000 × g for 1 minute at room temperature.
3. Repeat the wash step with 500 µL Wash Buffer (W5) with ethanol.
4. Discard the flow through and place the Spin Cartridge into the Wash Tube supplied with the kit.
5. Centrifuge the Spin Cartridge at maximum speed for 2-3 minutes at room temperature to remove any residual Wash Buffer. Discard the Wash Tube.
6. Proceed to the Elution Step, below.

Elution atep

1. Place the Spin Cartridge in a clean 1.7-mL Recovery Tube supplied with the kit.
2. Add 50-100 µL of Sterile, RNase-free water (pH >7.0) to the center of the Spin Cartridge.
3. Incubate at room temperature for 1 minute.
4. Centrifuge the Spin Cartridge at maximum speed for 1 minute at room temperature.
5. The Recovery Tube contains purified small RNA molecules. Remove and discard the cartridge. Based on the volume of elution buffer used for elution, the recovery of the elution volume will vary and is usually 90% of the elution buffer volume used.
6. Store small RNA at -80°C or use small RNA for the desired downstream application. You may determine the quality and quantity of the purified small RNA molecules as described on next.

RNA yield

The quantity of the purified small RNA molecules is easilyquantitated using UV absorbance at 260 nm or Quant-iT RNA Assay Kit.

UV Absorbance

1. Dilute an aliquot of the small sample in 10 mM Tris-HCl, pH 7.0. Mix well. Transfer to a cuvette (1-cm path length).  Note: The RNA must be in a neutral pH buffer to accurately measure the UV absorbance.
2. Determine the OD260 of the solution using a spectrophotometer blanked against 10 mM Tris-HCl, pH 7.0. Calculate the amount of total RNA using the following formula: Total RNA (μg) = OD260 x 40 μg/(1 OD260x 1 mL) x dilution factor x total sample volume (mL)

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.

RNA quality

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.

Results

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.

Expected yields

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.

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1. Elbashir SM, Harborth, J, Weber K, and Tuschl T (2002). Analysis of Gene Function in Somatic Mammalian Cells Using Small Interfering RNAs. Methods 26, 199–213.
2. Lim LP, Glasner ME, Yekta S, Burge CB, Bartel DP (2003). Vertebrate microRNA Genes. Science 299, 1540.
3. Liu CG, Calin GA, Meloon B, et.al. (2004). An Oligonucleotide Microchip for Genome-wide microRNA Profiling in Human and Mouse Tissues. Proc. Natl. Acad. Sci. USA 101, 9740–9744.

25-0753   Version C     18-Jul-2005