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The RiboMinus™ Transcriptome Isolation Kits provide a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large ribososmal RNA (rRNA) from total RNA for transcriptome analysis. The purification method is not dependent on the polyadenylation status or presence of a 5′-cap structure on the RNA. See below for more details on the purification protocol.
The isolation of RNA fraction depleted of ribososmal RNA is achieved by the selective removal of the large rRNA molecules from total RNA. The resulting rRNA depleted RNA fraction is termed as RiboMinus™ RNA Using the kit to isolate RiboMinus™ RNA results in efficient (>98%) removal of large rRNA molecules (yeast: 18S and 25/26S; bacteria 16S and 23S) from 10 μg total RNA enabling the analysis of the whole transcriptome without any interference from rRNA that account for ~90-95% RNA species in total RNA.
System Overview
The RiboMinus™ Transcriptome Isolation Kits are based on the selective removal of abundant large ribosomal RNA molecules from total RNA and concentrating the RiboMinus™ RNA enriched fraction. Total RNA is hybridized with species-specific, rRNA sequence-specific 5’-biotin labeled oligonucleotide probes (RiboMinus™ Yeast Probes or RiboMinus™ Bacteria Probes). The rRNA/5’-biotin labeled probe complex is removed from the sample with streptavidin coated magnetic beads (RiboMinus™ Magnetic Beads). The RiboMinus™ RNA sample is then concentrated using RiboMinus™ Concentration Module for a spin column-based centrifugation protocol or using ethanol precipitation. The binding conditions of the spin column method are optimized for the RiboMinus™ RNA sample with ethanol and Binding Buffer (L3). The sample is loaded onto a spin column. The RiboMinus™ RNA binds to the silica-based membrane in the column and impurities are removed by thorough washing with Wash Buffer (W5). The RNA is then eluted in sterile RNase free water.
Advantages
Using the RiboMinus™ Transcriptome Isolation Kits to isolate RiboMinus™ RNA (rRNA depleted RNA) provides the following advantages:
RiboMinus™ RNA
The large ribososmal RNA depleted RNA fraction is termed as RiboMinus™ RNA fraction. The RiboMinus™ RNA fraction contains polyadenylated (polyA) mRNA (yeast), non-polyadenylated RNA, pre-processed RNA, tRNA, smal rRNAs (5S rRNA, and additional 5.8S rRNA for eucaryotic RNA), and may also contain regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA), snRNA, and other RNA transcripts of yet unknown function.
The RiboMinus™ RNA molecules are part of the transcriptome and are important in protein coding, signaling, structural support of subcellular elements, and transcriptional/post transcriptional regulation. The transcriptome is defined as the complete collection of transcribed elements of the genome (Ruan et al., 2004) and contains mRN transcripts and non-mRNA transcripts including RiboMinus™ RNA. Transcriptome analysis is gaining increased attention in gene expression analysis. Since large rRNA constitutes 90-95% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult and suggests the need for developing procedures for transcriptome isolation.
Drawbacks of RNA Purification Methods
Current methods for RNA purification do not allow for efficient isolation of transcriptome. The total RNA purification methods result in enriching the large rRNA molecules while the mRNA purification methods use polyA-selection and/or cap-binding approaches (for eucaryotic RNA) that do not enrich the complete transcriptome.
The RiboMinus™ Transcriptome Isolation Kits offer a novel method of isolating transcriptome and involves selective removal of large rRNA from total RNA. The isolated transcriptome is >98% depleted in rRNA and is enriched in all RNA transcripts of interest enabling whole transcriptome analysis.
Downstream Applications
The isolated RiboMinus™ RNA is suitable for use in downstream applications such as microarray analysis, qRT-PCR, and cDNA library construction.
The isolation of RNA fraction depleted of ribososmal RNA is achieved by the selective removal of the large rRNA molecules from total RNA. The resulting rRNA depleted RNA fraction is termed as RiboMinus™ RNA Using the kit to isolate RiboMinus™ RNA results in efficient (>98%) removal of large rRNA molecules (yeast: 18S and 25/26S; bacteria 16S and 23S) from 10 μg total RNA enabling the analysis of the whole transcriptome without any interference from rRNA that account for ~90-95% RNA species in total RNA.
System Overview
The RiboMinus™ Transcriptome Isolation Kits are based on the selective removal of abundant large ribosomal RNA molecules from total RNA and concentrating the RiboMinus™ RNA enriched fraction. Total RNA is hybridized with species-specific, rRNA sequence-specific 5’-biotin labeled oligonucleotide probes (RiboMinus™ Yeast Probes or RiboMinus™ Bacteria Probes). The rRNA/5’-biotin labeled probe complex is removed from the sample with streptavidin coated magnetic beads (RiboMinus™ Magnetic Beads). The RiboMinus™ RNA sample is then concentrated using RiboMinus™ Concentration Module for a spin column-based centrifugation protocol or using ethanol precipitation. The binding conditions of the spin column method are optimized for the RiboMinus™ RNA sample with ethanol and Binding Buffer (L3). The sample is loaded onto a spin column. The RiboMinus™ RNA binds to the silica-based membrane in the column and impurities are removed by thorough washing with Wash Buffer (W5). The RNA is then eluted in sterile RNase free water.
Advantages
Using the RiboMinus™ Transcriptome Isolation Kits to isolate RiboMinus™ RNA (rRNA depleted RNA) provides the following advantages:
- Rapid and efficient isolation of high-quality RiboMinus™ RNA using probes specific to the large rRNA species
- Specifically designed to isolate RiboMinus™ RNA enriched in (polyA) mRNA (yeast), non-polyadenylated RNA, pre-processed RNA, tRNA, and small rRNAs
- Minimal contamination from rRNA molecules
- Reliable performance of the RiboMinus™ RNA in downstream applications such as microarray analysis, cDNA library construction, and qRT-PCR
RiboMinus™ RNA
The large ribososmal RNA depleted RNA fraction is termed as RiboMinus™ RNA fraction. The RiboMinus™ RNA fraction contains polyadenylated (polyA) mRNA (yeast), non-polyadenylated RNA, pre-processed RNA, tRNA, smal rRNAs (5S rRNA, and additional 5.8S rRNA for eucaryotic RNA), and may also contain regulatory RNA molecules such as microRNA (miRNA) and short interfering RNA (siRNA), snRNA, and other RNA transcripts of yet unknown function.
The RiboMinus™ RNA molecules are part of the transcriptome and are important in protein coding, signaling, structural support of subcellular elements, and transcriptional/post transcriptional regulation. The transcriptome is defined as the complete collection of transcribed elements of the genome (Ruan et al., 2004) and contains mRN transcripts and non-mRNA transcripts including RiboMinus™ RNA. Transcriptome analysis is gaining increased attention in gene expression analysis. Since large rRNA constitutes 90-95% RNA species in total RNA, whole transcriptome analysis without any contamination from rRNA is very difficult and suggests the need for developing procedures for transcriptome isolation.
Drawbacks of RNA Purification Methods
Current methods for RNA purification do not allow for efficient isolation of transcriptome. The total RNA purification methods result in enriching the large rRNA molecules while the mRNA purification methods use polyA-selection and/or cap-binding approaches (for eucaryotic RNA) that do not enrich the complete transcriptome.
The RiboMinus™ Transcriptome Isolation Kits offer a novel method of isolating transcriptome and involves selective removal of large rRNA from total RNA. The isolated transcriptome is >98% depleted in rRNA and is enriched in all RNA transcripts of interest enabling whole transcriptome analysis.
Downstream Applications
The isolated RiboMinus™ RNA is suitable for use in downstream applications such as microarray analysis, qRT-PCR, and cDNA library construction.
Introduction
This quick reference protocol is included for experienced users of the RiboMinus™ Yeast Transcriptome Isolation Kit and RiboMinus™ Bacteria Transcriptome Isolation Kit. If you are a first time user, follow the detailed protocol in this manual.
This quick reference protocol is included for experienced users of the RiboMinus™ Yeast Transcriptome Isolation Kit and RiboMinus™ Bacteria Transcriptome Isolation Kit. If you are a first time user, follow the detailed protocol in this manual.
| Step | Action |
|---|---|
| Isolating Total RNA | Isolate high-quality total RNA from yeast or bacteria cells using a method of choice prior to using these kits. You will need 2-10 μg total RNA in <20 μl per reaction. |
| Preparing RiboMinus™ Magnetic Beads | 1. Resuspend the RiboMinus™ Magnetic Beads in its bottle by thorough vortexing. 2. Pipet 250 μl of the bead suspension into a sterile, RNase-free, 1.5-ml microcentrifuge tube. 3. Place the tube with the bead suspension on a magnetic stand for 1 minute. Gently aspirate and discard the supernatant. 4. Add 250 μl sterile, RNase-Free Water to the beads and resuspend beads. Place the tube on a magnetic stand for 1 minute. Gently aspirate and discard the supernatant. 5. Repeat Step 4 once. 6. Resuspend beads in 250 μl Hybridization Buffer (B10). Place the tube on a magnetic stand for 1 minute. Gently aspirate and discard the supernatant. 7. Resuspend beads in 100 μl Hybridization Buffer (B10) and keep the beads at room temperature until use. |
| Selective Hybridization | Perform hybridization of your total RNA sample with the RiboMinus™ Yeast Probe or RiboMinus™ Bacteria Probe, depending on the source of total RNA as below. 1. To a sterile, RNase-free 1.5 ml microcentrifuge tube, add: Total RNA (2-10 μg): <20 μl RiboMinus™ Probe (100 pmol/μl): 4 μl Hybridization Buffer (B10): 100 μl 2. Incubate the tube at 37°C for 5 minutes to denature RNA. 3. Place the sample on ice for at least 30 seconds 4. Proceed to Removing rRNA. |
| Removing rRNA | 1. Briefly centrifuge the tube with the cooled hybridized sample (from Step 3), above to collect the sample to the bottom of the tube. 2. Transfer ~124 μl of the cooled hybridized sample (from Step 3, above) to the prepared RiboMinus™ Magnetic beads from Step 7, above, and mix well by vortexing. 3. Incubate the tube at 37°C for 15 minutes. During incubation, gently mix the contents occasionally. 4. Place the tube on a magnetic stand for 1 minute to pellet the rRNA-probe complex. The supernatant contains the RiboMinus™ RNA fraction. 5. Transfer the supernatant (~224 μl) to a tube capable of holding 3X volume of the supernatant. |
| Concentrating RiboMinus™ RNA using RiboMinus™ Concentration Module | See below for concentrating the RiboMinus™ RNA using the RiboMinus™ Concentration Module or using ethanol precipitation. 1. To the sample from Step 5, above, add 250 μl Binding Buffer (L3) and 125 μl 96-100% ethanol. Mix well by vortexing. 2. Bind the sample from Step 1 containing Binding Buffer (L3) and ethanol to the spin column. Centrifuge the column at 12,000 × g for 1 minute at room temperature. Discard the flow through. 3. Wash the column with 200 μl Wash Buffer (W5) with ethanol. Centrifuge the column at ≥12,000 × g for 1 minute at room temperature. Discard the flow through. 4. Repeat the wash step once. 5. Discard the tube and place the column into a clean Wash Tube, supplied with the kit. 6. Centrifuge the column at ≥12,000 × g for 2–3 minutes at room temperature to remove any residual Wash Buffer (W5). Place the column in a 1.5-ml Recovery Tube. 7. Elute with 10-15 μl of RNase-Free Water. Incubate the column at room temperature for 1 minute. Centrifuge the column at ≥12,000 × g for 1 minute. The Recovery Tube contains purified RiboMinus™ RNA. 8. Store RiboMinus™ RNA at -80°C or place RiboMinus™ RNA on ice to proceed to desired downstream application. |
System Specifications
Starting Material: 2-10 μg total RNA in <20 μl
rRNA Removal: >98%
RiboMinus™ RNA Yield: ≥0.5 μg from 10 μg total RNA
RiboMinus™ Probes Specifications
RiboMinus™ Yeast Probes:
Probe Contents: 2 probes each for 18S and 25/26S rRNA
Probe Specificity: Yeast
Probe Size: 18-21 oligonucleotides
Probe Label: 5′-biotin label
LNA™ Content: Each probe contains 5-7 LNA™ monomers in the oligonucleotide
Probe Mixture Concentration: 100 pmol/μl
RiboMinus™ Bacteria Probes:
Probe Contents: 2 probes each for 16S and 23S rRNA
Probe Specificity: Bacteria
Probe Size: 19-20 oligonucleotides
Probe Label: 5′-biotin label
LNA™ Content: Each probe contains 4-5 LNA™ monomers in the oligonucleotide
Probe Mixture Concentration: 100 pmol/μl
RiboMinus™ Magnetic Bead Specifications
The RiboMinus™ Magnetic Beads are streptavidin-coated magnetic beads.
Bead Binding Capacity: >2500 pmoles free biotin per mg RiboMinus™ Magnetic Beads
Bead Size: 1 μm diameter
Magnet Particle: Superparamagnetic polydisperse core-shell polystyrene particles
Concentration: 12 mg/ml
Specific Gravity: 1.1-1.4 g/cm3
RiboMinus™ Concentration Module Specifications
Binding Capacity: ~5 μg nucleic acid
Column Reservoir Capacity: 700 μl
Wash Tube Capacity: 2.0 ml
Recovery Tube Capacity: 1.5 ml
Centrifuge Compatibility: Capable of centrifuging at >10,000 x g
Elution Volume: 10-15 μl
TOPStarting Material: 2-10 μg total RNA in <20 μl
rRNA Removal: >98%
RiboMinus™ RNA Yield: ≥0.5 μg from 10 μg total RNA
RiboMinus™ Probes Specifications
RiboMinus™ Yeast Probes:
Probe Contents: 2 probes each for 18S and 25/26S rRNA
Probe Specificity: Yeast
Probe Size: 18-21 oligonucleotides
Probe Label: 5′-biotin label
LNA™ Content: Each probe contains 5-7 LNA™ monomers in the oligonucleotide
Probe Mixture Concentration: 100 pmol/μl
RiboMinus™ Bacteria Probes:
Probe Contents: 2 probes each for 16S and 23S rRNA
Probe Specificity: Bacteria
Probe Size: 19-20 oligonucleotides
Probe Label: 5′-biotin label
LNA™ Content: Each probe contains 4-5 LNA™ monomers in the oligonucleotide
Probe Mixture Concentration: 100 pmol/μl
RiboMinus™ Magnetic Bead Specifications
The RiboMinus™ Magnetic Beads are streptavidin-coated magnetic beads.
Bead Binding Capacity: >2500 pmoles free biotin per mg RiboMinus™ Magnetic Beads
Bead Size: 1 μm diameter
Magnet Particle: Superparamagnetic polydisperse core-shell polystyrene particles
Concentration: 12 mg/ml
Specific Gravity: 1.1-1.4 g/cm3
RiboMinus™ Concentration Module Specifications
Binding Capacity: ~5 μg nucleic acid
Column Reservoir Capacity: 700 μl
Wash Tube Capacity: 2.0 ml
Recovery Tube Capacity: 1.5 ml
Centrifuge Compatibility: Capable of centrifuging at >10,000 x g
Elution Volume: 10-15 μl
You will need to isolate high-quality total RNA from cells or tissues using a method of choice prior to using this kit. To obtain high-quality total RNA, follow the guidelines recommended below.
General Handling of RNA
Observe the following guidelines to prevent RNase contamination:
Total RNA Isolation
Total RNA can be isolated from tissue or cells using the method of choice. We recommend isolating total RNA using the Micro-to-Midi™ Total RNA Purification System or TRIzol® Reagent available from Invitrogen.
You will use 2-10 μg total RNA in less than 20 μl for each reaction. Resuspend isolated total RNA in DEPC-treate water accordingly (≥0.5 μg/μl).
Check the quality of your total RNA, including DNA contamination (see below). Store your total RNA at -80°C and avoid repeated freezing and thawing of total RNA.
If your downstream application requires DNA-free RNA, perform DNase-treatment of the total RNA before purifying RiboMinus™ RNA.
Checking the Total RNA Quality
To check total RNA integrity, analyze ∼0.5 μg of your RNA by agarose/ethidium bromide gel electrophoresis. You should see the following on an agarose gel:
General Handling of RNA
Observe the following guidelines to prevent RNase contamination:
- Use disposable, individually wrapped, sterile plasticware
- 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 o the skin
- Always use proper microbiological aseptic techniques when working with RNA
- Use RNase AWAY® Reagent to remove RNase contamination from surfaces
Total RNA Isolation
Total RNA can be isolated from tissue or cells using the method of choice. We recommend isolating total RNA using the Micro-to-Midi™ Total RNA Purification System or TRIzol® Reagent available from Invitrogen.
You will use 2-10 μg total RNA in less than 20 μl for each reaction. Resuspend isolated total RNA in DEPC-treate water accordingly (≥0.5 μg/μl).
Check the quality of your total RNA, including DNA contamination (see below). Store your total RNA at -80°C and avoid repeated freezing and thawing of total RNA.
If your downstream application requires DNA-free RNA, perform DNase-treatment of the total RNA before purifying RiboMinus™ RNA.
Checking the Total RNA Quality
To check total RNA integrity, analyze ∼0.5 μg of your RNA by agarose/ethidium bromide gel electrophoresis. You should see the following on an agarose gel:
- for yeast total RNA: 18S rRNA band (∼2.0 kb) and 25/26S rRNA band (∼3.8 kb)
- for bacterial total RNA: 16S rRNA band (∼1.5 kb) and 23S rRNA band (∼2.9 kb)
- The larger bands should be approximately twice the intensity of the smaller bands
Introduction
Instructions are provided in this section for selective hybridization of rRNA to the RiboMinus™ Probe and removal of rRNA using RiboMinus™ Magnetic Beads.
RiboMinus™ Probes
The RiboMinus™ Yeast Probes and RiboMinus™ Bacteria Probes are mixtures of oligonucleotide probes containing 2 probes each specific for 18S rRNA and 25/26S rRNA (yeast) or 16S rRNA and 23S rRNA (bacteria), respectively. The probes are designed to hybridize species-specific with highly conserved regions of the large rRNA molecules.
Each probe is single-stranded and contains 4-7 LNA™ (Locked Nucleic Acid) monomers incorporated at specific locations. The incorporation of LNA™ into the oligonucleotide probe increases the depletion efficiency of the rRNA from the samples without increasing the amount of beads or probe concentration.
The 5′-end of each probe is conjugated to biotin to allow removal of rRNA/probe complexes by binding to stretptavidin RiboMinus™ Magnetic Beads
RiboMinus™ Magnetic Beads
The RiboMinus™ Magnetic Beads are streptavidin-coated magnetic beads used for the removal of probe/rRNA complexes from the sample. The beads bind to the biotin-labeled probe complexed with rRNA or the probe alone. The beads are 1 μm polystyrene beads with a magnetic core that is strong enough to separate the bound complex from the solvent in a short period of time. The beads do not promote non-specific binding of any other RNA molecules. The size and the biotin binding capacity of the RiboMinus™ Magnetic Beads is optimized for use with this kit and results in >98% depletion of rRNA using 10 μg total RNA as the starting material. Avoid using any other streptavidin-coated magnetic beads with this kit.
Materials Needed
You will need the following items:
- Total RNA
- Magna-Sep™ Magnetic Particle Separator or equivalent
- RNase-free microcentrifuge tubes
- Water baths or heat blocks set to 70-75°C and 37°C
- Ice
Components supplied with the kit
- RiboMinus™ Magnetic Beads, keep on ice until use
- RiboMinus™ Yeast Probe or RiboMinus™ Bacteria Probe, keep on ice until use
- Hybridization Buffer (B10)
- RNase-Free Water
Follow the recommendations for handling the RiboMinus™ Magnetic Beads below for best results:
- During the mixing and washing steps of the magnetic beads, mix beads using a vortex. A low speed centrifuge pulse may be required to remove beads stuck in the tube cap. Avoid mixing by pipetting up and down as it results in bead loss.
- Do not allow the beads to dry as drying reduces the bead efficiency. During all washing steps with beads, add water or buffer to the tube containing beads while the tube is still on a magnetic stand to prevent drying of beads. Remove the tube from the magnet and resuspend the beads as described above.
- To aspirate the supernatant after bead washing, place the pipette tip at the opposite side of the tube, away from the beads. Carefully remove the supernatant without disturbing or removing any beads.
- Do not submerge the magnetic stand in water. To clean the magnetic stand, spray the stand with ethanol and wipe it with a paper towel.
The RiboMinus™ Hybridization Buffer (B10) contains 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.
Preparing RiboMinus™ Magnetic Beads
Follow the recommendations for handling beads and performing the washing steps.
- Resuspend the RiboMinus™ Magnetic Beads in its bottle by thoroughly vortexing.
- Pipet 250 μl of the bead suspension into a sterile, RNase-free, 1.5 ml microcentrifuge tube.
- Place the tube with the bead suspension on a magnetic separator for 1 minute. The beads will settle to the sid of the tube that faces the magnet. Aspirate and discard the supernatant.
- Add 250 μl sterile, RNase-Free Water supplied with the kit to the beads and resuspend beads by vortexing.
- Place the tube on a magnetic separator for 1 minute. Gently aspirate and discard the supernatant.
- Repeat Steps 4-5 once.
- Resuspend beads in 250 μl Hybridization Buffer (B10). Place the tube on a magnetic separator for 1 minute. Gently aspirate and discard the supernatant.
- Resuspend beads in 100 μl Hybridization Buffer (B10) and keep the beads at 37°C until use
Continue to Hybridization Step
Hybridization Step
Instructions are provided below to perform hybridization for 2-10 μg of your total RNA sample with the RiboMinus Yeast Probe or RiboMinus™ Yeast Probe, depending o the species of the total RNA. If you wish to process >10 μg total RNA sample, divide your sample into two samples, each containing <10 μg total RNA.
- Set a water bath or heat block to 37°C.
- To a sterile, RNase-free 1.5 ml microcentrifuge tube, add the following:
Total RNA (2-10 μg): <20 μl
RiboMinus™ Probe (100 pmol/μl): 4 μl
Hybridization Buffer (B10): 100 μl - Incubate the tube at 37°C for 5 minutes to denature RNA.
- Place the sample on ice for at least 30 seconds.
- Proceed to Removing rRNA
Removing rRNA
- Set a water bath or heat block to 37°C.
- Briefly centrifuge the tube with the cooled hybridized sample (from Step 4, above) to collect the sample to the bottom of the tube.
- Transfer the sample (~124 μl) to the prepared RiboMinus™ Magnetic beads from Step 8, above. Mix well by vortexing the tube repeatedly.
- Incubate the tube at 37°C for 15 minutes. During incubation, gently mix the contents occasionally. Briefly centrifuge the tube to collect the sample to the bottom of the tube.
- Place the tube on a magnetic separator for 1 minute to pellet the rRNA-probe complex. Do not discard the supernatant. The supernatant contains RiboMinus™ RNA.
- Transfer the supernatant (~224 μl) to a tube capable of holding 3X the volume of the supernatant.
You can concentrate the RiboMinus™ RNA using RiboMinus™Concentration Module with a rapid and optimized spin-based protocolor using ethanol precipitation.
Introduction
The RiboMinus™ Concentration Module is designed for concentrating RiboMinus™ RNA using spin column-based centrifugation protocol in a total time of 10-15 minutes.
Materials Needed
Components supplied with the RiboMinus™ Concentration Module
The RiboMinus™ Concentration Module Binding Buffer (L3) contains 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.
Before Starting
Add 6 ml 96-100% ethanol to 1.5 ml Wash Buffer (W5) included with the kit. Store the Wash Buffer (W5) with ethanol at room temperature.
Binding Step
Washing Step
Elution Step
The RiboMinus™ Concentration Module is designed for concentrating RiboMinus™ RNA using spin column-based centrifugation protocol in a total time of 10-15 minutes.
Materials Needed
- RiboMinus™ RNA sample from Step 6
- 96-100% ethanol
- Microcentrifuge capable of centrifuging >12,000 x g
Components supplied with the RiboMinus™ Concentration Module
- Binding Buffer (L3)
- Wash Buffer (W5)
- RNase-Free Water
- Spin Column with Collection Tubes
- Wash Tubes
- Recovery Tubes
The RiboMinus™ Concentration Module Binding Buffer (L3) contains 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.
Before Starting
Add 6 ml 96-100% ethanol to 1.5 ml Wash Buffer (W5) included with the kit. Store the Wash Buffer (W5) with ethanol at room temperature.
Binding Step
- To the sample from Step 6, add 250 μl Binding Buffer (L3) and 125 μl 96-100% ethanol. Mix well by vortexing.
- Load the sample containing Binding Buffer (L3) and ethanol to the column.
- Centrifuge the column at ≥12,000 × g for 1 minute at room temperature. Discard the flow through.
- Proceed to Washing Step
Washing Step
- Add 200 μl Wash Buffer (W5) with ethanol to the column
- Centrifuge the column at ≥12,000 × g for 1 minute at room temperature. Discard the flow through.
- Repeat the wash step with 200 μl Wash Buffer (W5) with ethanol.
- Discard the collection tube and place the column into a clean Wash Tube supplied with the kit.
- Centrifuge the column at maximum speed for 2-3 minutes at room temperature to remove any residual Wash Buffer (W5). Discard the Wash Tube
- Proceed to Elution Step, below.
Elution Step
- Place the Spin Column in a clean 1.5-ml Recovery Tube supplied with the kit.
- Add 10-15 μl of RNase-Free Water to the center of the column. Incubate the column at room temperature for 1 minute.
- Centrifuge the column at maximum speed for 1 minute at room temperature. The Recovery tube contains purified and concentrated RiboMinus™ RNA sample that is depleted of rRNA.
- Place RiboMinus™ RNA on ice to proceed to desired downstream application or store RiboMinus™ RNA at -80°C until further use.
Introduction
This section includes a protocol for Ethanol Precipitation to further concentrate the RiboMinus™ RNA. You will need at least 1 hour to perform the ethanol precipitation.
Materials Needed
Ethanol Precipitation
RNA Yield
The quantity of the purified RiboMinus™ RNA is easily quantitated using UV absorbance at 260 nm or Quant-iT™ RNA Assay Kit.
UV Absorbance
Total RNA (μg) = OD260 x 40 μg/(1 OD260 x 1 ml) x dilution factor x total sample volume (ml)
The typical yield of RNA using the RiboMinus™ Transcriptome Isolation Kits (Yeast and Bacteria) is ~0.5 μg RNA from 10 μg total RNA sample.
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
The RNA isolated using the RiboMinus™ Transcriptome Isolation Kit is of high-quality and is >98% depleted in rRNA species. To verify rRNA depletion, perform agarose gel electrophoresis of the sample or use a bioanalyzer (see below for examples).
Gel Electro-phoresis to assess RNA Quality
You can use agarose gel electrophoresis analysis to verifyl depletion of the large rRNA bands as compared to a control sample and absence of contaminating DNA and RNA degradation.
Bioanalyzer to assess RiboMinus™ RNA Quality
The efficiency of rRNA depletion in RiboMinus™ RNA, RNA degradation, and RNA concentration can be analyzed using a bioanalyzer such as the Agilent 2100 bioanalyzer with an RNA LabChip®.
This section includes a protocol for Ethanol Precipitation to further concentrate the RiboMinus™ RNA. You will need at least 1 hour to perform the ethanol precipitation.
Materials Needed
- RiboMinus™ RNA sample (Step 7)
- Glycogen, 20 μg/μl
- 3 M sodium acetate in RNAse-free water
- 96-100% cold ethanol
- 70% cold ethanol
- RNase-free water
- Sterile, RNase-free microcentrifuge tubes
- Microcentrifuge capable of centrifuging >12,000 x g
Ethanol Precipitation
- Add the following components to the RiboMinus™ RNA:
- 1 μl glycogen (20 μg/μl)
- 1/10th sample volume of 3 M sodium acetate
- 2.5X sample volumes of 100% ethanol
- Mix well and incubate at -20 or -80°C for a minimum of 30 minutes.
- Centrifuge the tube for 15 minutes ≥12,000 × g at 4°C.
- Carefully discard the supernatant without disturbing the pellet.
- Add 500 μl 70% cold ethanol.
- Centrifuge the tube for 5 minutes at ≥12,000 × g at 4°C.
- Carefully discard the supernatant without disturbing the pellet.
- Repeat Steps 6-8 once.
- Air-dry the pellet for ∼5 minutes; do not dry completely
- Resuspend the RNA pellet in ∼10-30 μl RNase-free water.
- Place RiboMinus™ RNA on ice to proceed to desired downstream application or store RiboMinus™ RNA at -80°C until further use.
RNA Yield
The quantity of the purified RiboMinus™ RNA is easily quantitated using UV absorbance at 260 nm or Quant-iT™ RNA Assay Kit.
UV Absorbance
- 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.
- 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 OD260 x 1 ml) x dilution factor x total sample volume (ml)
The typical yield of RNA using the RiboMinus™ Transcriptome Isolation Kits (Yeast and Bacteria) is ~0.5 μg RNA from 10 μg total RNA sample.
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
The RNA isolated using the RiboMinus™ Transcriptome Isolation Kit is of high-quality and is >98% depleted in rRNA species. To verify rRNA depletion, perform agarose gel electrophoresis of the sample or use a bioanalyzer (see below for examples).
Gel Electro-phoresis to assess RNA Quality
You can use agarose gel electrophoresis analysis to verifyl depletion of the large rRNA bands as compared to a control sample and absence of contaminating DNA and RNA degradation.
Bioanalyzer to assess RiboMinus™ RNA Quality
The efficiency of rRNA depletion in RiboMinus™ RNA, RNA degradation, and RNA concentration can be analyzed using a bioanalyzer such as the Agilent 2100 bioanalyzer with an RNA LabChip®.
Review the table below to troubleshoot problems that you may encounter using the RiboMinus™ Transcriptome Isolation Kits.
| Problem | Cause | Solution |
|---|---|---|
| Low RNA yield | Low RNA content | Various tissues have different RNA content and the yield is dependent on the sample. |
| Incorrect binding conditions when using the RiboMinus™ Concentration Module | For efficient binding of RiboMinus™ RNA to the spin column, always add 250 μl Binding Buffer (L3) and 125 μl 96-100% ethanol to the hybridized sample and mix well by vortexing prio | |
| Ethanol not added to Wash Buffer (W5) | Be sure to add 96–100% ethanol to Wash Buffer (W5) | |
| Incorrect elution conditions | Add water to the center of the column and perform incubation for 1 minute with water before centrifugation. RNA quantitation performed with water Be sure the RNA quantitation using | |
| 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 | |
| Loss of pellet during to ethanol precipitation | Remove supernatant from RNA pellet carefully. Use RiboMinus™ Concentration Module to concentrate the RiboMinus™ RNA. | |
| Incomplete removal of rRNA | Too much total RNA used | The protocols in this manual are designed to purify RiboMinus™ RNA from 2-10 μg total RNA. If you are using more than 10 μg total RNA, divide the sample into two sample aliquots, each containing <10 μg total RNA for RiboMinus™ RNA purification. |
| Low amount of magnetic beads or probe used | Be sure to use the recommended amounts of RiboMinus™ Probe and RiboMinus™ Magnetic Beads for efficient removal of rRNA. | |
| Incomplete removal of rRNA | Improper handling or drying of beads | To obtain the best results with RiboMinus™ Magnetic Beads, follow the guidelines for washing and mixing the beads, and aspirating the supernatant. Do not allow the beads to dry as drying reduces the bead efficiency. |
| RNA degraded | RNA contaminated with RNase | Follow the guidelines to prevent RNase contamination. |
| Poor quality starting materials | Always use fresh samples or samples frozen at -80°C for isolation of total RNA. Be sure to check the quality of your total RNA prior to use. | |
| Genomic DNA contamination | Total RNA contained genomic DNA | Perform DNase I digestion with the total RNA sample to remove any genomic DNA contamination before performing RiboMinus™ RNA purification. |
| Inhibition of downstream enzymatic reactions | Presence of ethanol in purified RNA sample | Traces of ethanol from the RiboMinus™ Concentration Module Wash Buffer (W5) or from ethanol precipitation can inhibit downstream enzymatic reactions. To remove Wash Buffer (W5), discard Wash Buffer flow through from the collection tube. Reinsert the spin column into the collection tube and centrifuge the spin column at maximum speed for 2 minutes to completely dry the column. If performing ethanol precipitation, make sure that ethanol is evaporated before resuspending the RiboMinus™ RNA pellet in RNase-free water. |
- Braasch, D. A., and Corey, D. R. (2001). Locked Nucleic Acid (LNA): Fine-tuning the Recognition of DNA and RNA. Chem Biol. 1, 1-7.
- McTigue, P. M., Peterson, R. J., and Kahn, J. D. (2004). Sequence-dependent Thermodynamic Parameters for Locked Nucleic Acid (LNA)-DNA Duplex Formation. Biochemistry. 43, 5388-5405.
- Ruan, Y., Le Ber, P., Ng, H., and Liu, E. (2004). Interrogating the Transcriptome. Trends Biotechnol. 22, 23-30.
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