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Ribominus Transcriptome Isolation Kit (Yeast and Bacteria) - for efficient transcriptome enrichment by depleting large

互联网2013-11-13

1490

实验原理

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.

实验步骤

1. 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.

2. 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.

3. 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.

4. 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.

5. Concentrating RiboMinus™ RNA using RiboMinus™ Concentration Module

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.

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