Ribominus™ Transcriptome Isolation Kit (Yeast and Bacteria) - for efficient transcriptome enrichment by depleting large ribosomal RNA from yeast and bacteria total RNA

The RiboMinus™ Transcriptome Isolation Kits provide a novel and efficient method to isolate RNA molecules of the transcriptome devoid of large ribosomal 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 ribosomal 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 ribosomal 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.

• 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

• 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 streptavidin 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.

1. Resuspend the RiboMinus™ Magnetic Beads in its bottle by thoroughly 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 separator for 1 minute. The beads will settle to the sid  of the tube that faces the magnet. Aspirate and discard the supernatant.


4. Add 250 μl sterile, RNase-Free Water supplied with the kit to the beads and resuspend beads by vortexing.


5. Place the tube on a magnetic separator for 1 minute. Gently aspirate and discard the supernatant.


6. Repeat Steps 4-5 once.


7. Resuspend beads in 250 μl Hybridization Buffer (B10). Place the tube on a magnetic separator for 1 minute. Gently aspirate and discard the supernatant.


8. 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.

1. Set a water bath or heat block to 37°C.
   


2. 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
   


3. Incubate the tube at 37°C for 5 minutes to denature RNA.
   


4. Place the sample on ice for at least 30 seconds.


5. Proceed to Removing rRNA

Removing rRNA

1. Set a water bath or heat block to 37°C.


2. Briefly centrifuge the tube with the cooled hybridized sample (from Step 4, above) to collect the sample to the bottom of the tube.


3. Transfer the sample (~124 μl) to the prepared RiboMinus™ Magnetic beads from Step 8, above. Mix well by vortexing the tube repeatedly.


4. 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.


5. 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.


6. 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.

• RiboMinus™ RNA sample from Step 6
• 96-100% ethanol
• Microcentrifuge capable of centrifuging >12,000 x g

• Binding Buffer (L3)
• Wash Buffer (W5)
• RNase-Free Water
• Spin Column with Collection Tubes
• Wash Tubes
• Recovery Tubes

1. To the sample from Step 6, add 250 μl Binding Buffer (L3) and 125 μl 96-100% ethanol. Mix well by vortexing.


2. Load the sample containing Binding Buffer (L3) and ethanol to the column.


3. Centrifuge the column at ≥12,000 × g for 1 minute at room temperature. Discard the flow through.


4. Proceed to Washing Step

1. Add 200 μl Wash Buffer (W5) with ethanol to the column


2. Centrifuge the column at ≥12,000 × g for 1 minute at room temperature. Discard the flow through.


3. Repeat the wash step with 200 μl Wash Buffer (W5) with ethanol.


4. Discard the collection tube and place the column into a clean Wash Tube supplied with the kit.


5. Centrifuge the column at maximum speed for 2-3 minutes at room temperature to remove any residual Wash Buffer (W5). Discard the Wash Tube


6. Proceed to Elution Step, below.

1. Place the Spin Column in a clean 1.5-ml Recovery Tube supplied with the kit.


2. Add 10-15 μl of RNase-Free Water to the center of the column. Incubate the column at room temperature for 1 minute.


3. 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.


4. Place RiboMinus™ RNA on ice to proceed to desired downstream application or store RiboMinus™ RNA at -80°C until further use.

• 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

1. 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
   
   
2. Mix well and incubate at -20 or -80°C for a minimum of 30 minutes.
   


3. Centrifuge the tube for 15 minutes ≥12,000 × g at 4°C.
   


4. Carefully discard the supernatant without disturbing the pellet.
   


5. Add 500 μl 70% cold ethanol.
   


6. Centrifuge the tube for 5 minutes at ≥12,000 × g at 4°C.
   


7. Carefully discard the supernatant without disturbing the pellet.
   


8. Repeat Steps 6-8 once.
   


9. Air-dry the pellet for ∼5 minutes; do not dry completely
   


10. Resuspend the RNA pellet in ∼10-30 μl RNase-free water.
    


11. Place RiboMinus™ RNA on ice to proceed to desired downstream application or store RiboMinus™ RNA at -80°C until further use.

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:

1. 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.


2. 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.


3. Ruan, Y., Le Ber, P., Ng, H., and Liu, E. (2004). Interrogating the Transcriptome. Trends Biotechnol. 22, 23-30.